Agilent Technologies 6400 Series User manual

Agilent 6400 Series Triple Quadrupole
LC/MS System
Quick Start Guide
Where to find information 3
Getting Started 5
Step 1. Start the Data Acquisition software 6
Step 2. Prepare the LC modules 12
Step 3. Prepare the Triple Quad instrument 16
Step 4. Set up and run an acquisition method 20
Step 5. Review results with the Qualitative Analysis program 27
Step 6. Analyze data with the Quantitative Analysis program 27
Use this guide for your first steps with the Agilent 6400 Series Triple Quad LC/MS, and as a
road map for your user information.
What is the Agilent 6400 Series Triple Quad LC/MS system?
The Agilent Triple Quadrupole LC/MS is a liquid chromatograph triple quadrupole
mass spectrometer that performs MS/MS using three sets of parallel rods (in this
case, quadrupole, hexapole, quadrupole). The first quadrupole separates ions into
precursor ions that are fragmented in the hexapole into product ions, which are
separated by the second quadrupole. Often, two or more precursor ions and their
product ions are monitored in sequence in MRM (multiple reaction monitoring)
mode. You can monitor up to 4000 MRM transitions by using Dynamic MRM.
The Agilent 6490 and 6495 Triple Quadrupoles contain the iFunnel Technology
which provide more efficient sampling of ions into the mass spectrometer. The
iFunnel Technology includes the Dual Offset Ion Funnel and these other
technologies: the Agilent Jet Stream Technology, and shorter desolvation assembly
with Hexabore Capillary.

What’s New in Data Acquisition
2 Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide
The Agilent 6495, 6490 and 6460 Triple Quad LC/MS are shipped by default with
the Agilent Jet Stream Technology that utilizes a super-heated sheath gas to
collimate the nebulizer spray which dramatically increases the number of ions that
enter the mass spectrometer.
You can set up an Agilent Triple Quad LC/MS with the Agilent 1100, 1200, 1260 or
1290 Infinity LC modules.
Also, the Agilent Triple Quad LC/MS comes with Agilent MassHunter Workstation
Software that includes three major programs:
•Data Acquisition – From one screen you can tune the mass spectrometer,
control and monitor instrument parameters, set up acquisition methods and
worklists containing multiple samples and monitor real-time run plots.
•Quantitative Analysis – From one screen you can set up a batch of data files and
quantify, evaluate and requantify the results. From this screen you have access
to the Method Editor for setting up and editing the quantitation methods.
•Qualitative Analysis – From one screen you can extract and integrate
chromatograms, subtract background, extract peak spectra, and compare data
from different types of data files.
What’s New in Data Acquisition
The Data Acquisition program for the 6400 Series Triple Quadrupole has many
new features in this revision.
in B.07.00
•The supported models are 6420A, 6430A, 6460A, 6460C, 6490A, and 6495A.
•The supported LC models are 1100, 1200, 1260, 1290, HTS, and HTC.
•You can autotune the 6495 with the Agilent Jet Stream.
•Autotune is faster for the 6490 and the 6495 than in previous versions.
•The 6495 supports Unit, Wide, and Widest resolutions.
•Fast scan autotune is not supported for the 6495.
•Optimizer allows you to set the injection volume for a compound.
•Optimizer allows you to set the number of transitions to find during
optimization.
•Optimizer allows you to open a different database from the File menu in
Optimizer.

Where to find information
Help
Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide 3
Where to find information
Help
Press F1 To get more information about a pane, window or dialog box, place the
cursor on the part of the pane, window or dialog box of interest and press F1.
Help menu From the Help menu, access “How-to” help and reference help.
Documents
You can find these manuals delivered with the Triple Quad hardware or software.
You can also find a PDF version on the installation disk, in the Manuals folder.
Many of these manuals are also available at www.chem.agilent.com.
Installation Guide This guide is used to install the hardware and software,
configure the instrument, and verify performance.
Upgrade Guide This guide is used to upgrade your MassHunter Workstation
software from a previous version.
Maintenance Guide Use this guide to help maintain and troubleshoot your
Agilent Triple Quad LC/MS.
Concepts Guide - The Big Picture Learn the background information to help you
understand operation of the hardware and software.
Data Acquisition for 6400 Series Triple Quad Familiarization Guide Do the
exercises to learn to use the Triple Quad LC/MS hardware and Data Acquisition
program for 6400 Series Triple Quad.
Study Manager Quick Start Guide Use this guide to learn to use the MassHunter
Study Manager software.
MassHunter Optimizer Software Quick Start Guide Use this guide to learn about
the MassHunter Optimizer program. The MassHunter Optimizer program provides
a way to automatically optimize the data acquisition parameters for MRM mode
(multiple-reaction monitoring) on a Triple Quad instrument for each individual
compound analyzed.

Where to find information
Training
4 Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide
MassHunter QQQ Compliance Quick Start Guide Use this guide to learn about the
MassHunter QQQ Compliance program.
MassHunter Quant Compliance Quick Start Guide Use this guide to learn about
the MassHunter Quant Compliance program. (This guide is on the Quantitative
Analysis disk.)
Qualitative Analysis Familiarization Guide Do the exercises to learn to use the
Qualitative Analysis program. (This guide is on the Qualitative Analysis disk.)
For information on what is new in the Qualitative Analysis program, refer to the
Qualitative Analysis Familiarization Guide.
Quantitative Analysis Familiarization Guide Do the exercises to learn to use the
Quantitative Analysis program. (This guide is on the Quantitative Analysis disk.)
For information on what is new in the Quantitative Analysis program, refer to the
Quantitative Analysis Familiarization Guide.
Training
Familiarization Guide Use all three familiarization guides to get to know the
software.
Quick Start Guide Use the quick start guides for Study Manager, Optimizer and
Compliance to get to know these programs.
Training Courses Visit www.chem.agilent.com to view a listing of training courses
for the Agilent Triple Quad LC/MS.

Getting Started
Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide 5
Getting Started
Install the Triple Quadrupole LC/MS hardware and software
Use the Installation Guide to install the hardware and software and verify
performance.
Set up, run, and analyze samples
The roadmap below shows you the steps to set up and run a batch of samples from
start to finish. Follow the instructions on the next pages to get started and to learn
where to find the information to help you with each step in this roadmap.
Start
Here!
Set up, run
and analyze
samples
3Prepare the
Triple Quad
instrument.
1Start the
Data
Acquisition
software.
2Prepare the
LC modules.
4Set up and
run an
acquisition
method.
5Review
results with
Qualitative
Analysis.
6Analyze data
with
Quantitative
Analysis.

Getting Started
Step 1. Start the Data Acquisition software
6 Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide
Step 1. Start the Data Acquisition software
The instructions below include the following assumptions:
•The hardware and software are installed.
•The instrument is configured.
Use instructions in the Installation Guide to configure the instrument for the
first time and any time you change the LC configuration.
The LC modules and the Triple Quad MS are turned on, but the LC pump is not
running.
After installation, you see all of the Agilent MassHunter Workstation Software
icons on your Desktop. To start the Data Acquisition program, double-click
the Data Acquisition icon.
The Data Acquisition window appears.
NOTE When Data Acquisition opens, the software engines automatically
start. If you need to restart them, right-click the Acq System
Launcher icon in the system tray, and click Start Engines.
If you have recently changed LC modules, remember to configure the
instrument again. See the Installation Guide for instructions.

Getting Started
Step 1. Start the Data Acquisition software
Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide 7
Windows—where you do most of your work
When you first start the Data Acquisition program, the main window appears. You
do almost all of your work within the eight windows of this main window. These
windows provide the tools to set up acquisition methods, run samples
interactively or automatically, monitor instrument status, monitor runs and tune
the instrument.
Figure 1 Main window of the Data Acquisition software program
Instrument Status
window
Chromatogram Plot
window
Method Editor
window
Spectrum Plot
window
Actuals
window
The Sample Run and Worklist windows are tabbed here. These three windows are “sharing” this space. You
click the tab to switch to a different window.

Getting Started
Step 1. Start the Data Acquisition software
8 Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide
Show/hide the windows You can show one window at a time on the screen
or up to seven windows. You can never hide all of the windows. To show or
hide a window, you click the commands in the View menu. You can also hide
a window by clicking the X icon in the upper right corner of the window.
When you click a window, the title of the active window changes to a different
color. Press F1 to obtain help on the active window. You can also drag a
window border to resize the window. If you double-click the title of the
window, the window “floats” outside of the main window. You can double-click
the title bar again to “dock” the window. You can also float and dock the
window when you right-click the title of the window and click Floating.
Instrument Status window With this window you view the status of each
device configured with the instrument: Error, Not ready, Pre-run, Post-run,
Running, Injecting, Idle, Offline, or Standby. You also set non-method control
and configuration parameters for the LC devices and the MS instrument.
This window displays the current status of each device both as text and by its
color-coding:
Table 1 Color Coding in the Instrument Status Window
Color Status
Red Error
Yellow Not ready
Purple Pre-run, post-run, Waiting
Blue Running, injecting
Green Idle
Dark gray Offline
Light gray Standby (for example, lamps off)

Getting Started
Step 1. Start the Data Acquisition software
Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide 9
Actuals window With this window you view the current value of selected
instrument parameters. See “Set up to view real-time parameter values
(actuals).” on page 14 for more information.
Chromatogram Plot window With this window you monitor the chromatogram
plots in real time. These plots can be user-defined signals and/or instrument
parameters. You select the plots in the Chromatogram tab in the QQQ tab in
the Method Editor window.
Spectrum Plot window With this window you monitor the spectral plot in real
time.

Getting Started
Step 1. Start the Data Acquisition software
10 Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide
Method Editor window With this window you enter acquisition parameters for
the method. If you click the button, then you can see the tune values in the
Tune Parameters dialog box.
Sample Run window With this window you enter sample information to run
individual samples interactively, and you can start a single sample run. You can
also specify an Override DA Method and select either Both Acquisition and DA
or DA Only for the Method Type, and then Data Analysis is run as part of the
method.

Getting Started
Step 1. Start the Data Acquisition software
Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide 11
Worklist window With this window you enter sample information for multiple
samples. When you run the worklist, the samples are automatically run in the
order listed in the worklist. You can select whether to run Acquisition Only, to run
Both Acquisition and DA, or to run DA only by selecting one of these options for
the Part of method to run in the Worklist Run Parameters dialog box.
Tune window With this window you tune the mass spectrometer. You can use the
automatic tuning algorithms that are provided, or you can manually tune the
instrument. You have to switch to the Tune Context to see this window.
This section shows the
current progress in the
Autotune or Checktune.
During an Autotune or Checktune,
some of the parameters are
ramped to determine the best
value.
If you make changes in the Manual Tune tab, you can print a Checktune
and Autotune report when you click Create Tune Report.

Getting Started
Step 2. Prepare the LC modules
12 Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide
Step 2. Prepare the LC modules
Read and follow the instructions in the online Help for each of the tasks in
the checklist described on the following pages. While you condition or
equilibrate the column, you can tune the Triple Quad MS.
1Switch LC stream to Waste.
When you are not acquiring data, you switch the direction of the LC stream away
from the MS ion source and to waste.
If you have the LC connected to a VWD or DAD, you can still monitor the
fluctuations of the VWD or DAD real-time chromatogram before a run.
aRight-click the QQQ device in the Instrument Status window.
bClick LC > Waste.
2Purge the LC pump.
Follow the directions for purging the pump in the User Guide for your
pump.

Getting Started
Step 2. Prepare the LC modules
Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide 13
3Set up to condition or equilibrate the column.
aType LC parameters, and click the Apply button in the toolbar to download
them to the LC.
bRight-click an LC module in the Instrument Status window to change any
non-method control parameters, if necessary.
cMonitor the baseline and adjust the plot to make sure the column is
equilibrated and the baseline stable. (See step 4and step 5on
page 14.)

Getting Started
Step 2. Prepare the LC modules
14 Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide
4Set up to view real-time parameter values (actuals).
As you prepare for a run and during a run, you want to see the actual
values of the instrument parameters. You can do this in the Instrument
Status window.
aRight-click the Actuals list to see the Setup command.
bClick Setup to bring up the list of Actuals available for monitoring.
cAdd all the parameter values you intend to monitor, and click the OK
button. Parameters that you may want to monitor include MS values
(such as heater, and vacuum pressure) or LC values (such as binary
pump, column, DAD, etc.) You can set the background and text color for
each parameter. You can also set a range for the parameters which are
numbers. If the value of the parameter is not within the limits which
you entered, then the background of the parameter is set to red.

Getting Started
Step 2. Prepare the LC modules
Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide 15
5Set up real-time plot displays.
As you condition the column, you can set up the displays to monitor the
effluent.
•Right-click the chromatogram plot, and click Change.
In the Edit Signal Plot dialog box, you can select the type of display signal.

Getting Started
Step 3. Prepare the Triple Quad instrument
16 Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide
Step 3. Prepare the Triple Quad instrument
Do a Checktune, Autotune or Manual Tune
1From the Context list, click Tune.
You can see the Instrument Status window, the Actuals window and the Tune
window when you switch to the Tune context. Click Tune in the View menu if
the Tune window is not visible.
2Click Checktune to evaluate if the MS parameters are within the limits to
produce the specified mass accuracy and resolution. Checktune takes
approximately 3 minutes to run for each polarity.
If the current tune file was last tuned with the Fast Scan Autotune, then the
Checktune algorithm only checks MS2. If the current tune file was last tuned
with Autotune, then the Checktune algorithm checks both MS1 and MS2.
If the current tune file was last tuned with the Fast Scan Autotune, then the
Fast Scan check box is marked on the Manual Tune tab.
Do a Checktune regularly.
You can run a Checktune with the following sources: ESI, AJS ESI, MMI and
HPLC-Chip.
If Checktune passes, then skip to step 5.
If Checktune fails, then you can try using the Manual Tune tab to fix the
problem. See the next step.

Getting Started
Step 3. Prepare the Triple Quad instrument
Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide 17
3Try the following quick changes to get Checktune to pass.
aClick the Manual Tune tab.
bIf the failure occurred in MS1, click the MS1 tab. Select the Peak Width
based on which resolution failed in Checktune. You can select Unit, Wide,
or Widest. Then, click the Adjust Gain & Offset button.
cIf the failure occurred in MS2, click the MS2 tab. Select the Peak Width
based on which resolution failed in Checktune. Then, click the Adjust Gain
& Offset button.
dIf the Adjust Gain and Offset passes successfully, then save the autotune
file. Click Manual Tune. Then, click the Files tab, and click Save.
eClick the Autotune tab.
fClick the Checktune button.
If Checktune fails again, you must run an Autotune, which is described next.
4Click Autotune to tune the MS automatically (approximately 30 minutes for
each ionization mode for all models except the 6490 and the 6495. The 6490 and
6495 take approximately 25 minutes per ionization mode.). The system
automatically changes different tune parameters to tune the MS. You only do an
Autotune when it is necessary.
For all instruments except the 6490 and the 6495, you can run an Autotune
with the following sources: AJS ESI, MMI, and HPLC-Chip. With the 6490 and
6495, you can only run an Autotune with the AJS ESI source.
If Autotune fails, then you mark the Start from Factory Defaults check box.
Then, if you click Autotune, the instrument is tuned starting from the factory
defaults (approximately 30 minutes for each polarity for all models except the
6490 and the 6495. The 6490 and 6495 take approximately 25 minutes per
ionization mode.).

Getting Started
Step 3. Prepare the Triple Quad instrument
18 Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide
Checktune and Autotune reports are automatically generated after Autotune
completes successfully. If Autotune fails, no reports are printed. You can check
the progress box in the lower right side of the Autotune tab to see the reasons
why the tune failed. Then, you can either fix the problem, or call the Agilent
service engineer and provide this information.
If Autotune fails or you assess that the Triple Quadrupole MS needs custom
values entered for its tune parameters, you can manually tune the instrument.
If you cannot get the instrument to tune successfully, then please call your
Agilent service engineer.
5Optional. Click the Fast Scan Autotune Only button if you want to tune
for Fast Scan. You only do this step if the Autotune results are acceptable.
Fast Scan acquisition is only supported on the 6490 with two of the scan
types: MS2 Scan and Product Ion scan. Fast Scan autotune only tunes with
the Wide (1.2) resolution, Samples set to 1 (on the Manual Tune >
Acquisition Tab), and the step size must be 0.2. The scan time in the
Acquisition tab is generated automatically based on the step size and the
mass range.
Fast Scan acquisition is not supported for the 6495. Instead, you achieve the
faster scan rate by increasing the step size to 0.3 m/z.
6From the Context list, click Acquisition.
Switch LC stream to MS
•After you condition the column and tune the Triple Quad MS, you switch the LC
stream from Waste to MS. See “Switch LC stream to Waste.” on page 12 for
how to do this.

Getting Started
Step 3. Prepare the Triple Quad instrument
Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide 19
Monitor MS baseline and spectral displays
If you did not monitor the LC baseline with a VWD or DAD, make sure that the
Triple Quadrupole baseline is stable, and no spectra of interfering intensity
appear in the display.
If you did monitor the LC baseline with a VWD or DAD, you change back to the
default Triple Quad displays.
1Right-click the chromatogram plot, and click Change.
2Select the MS signal, and click OK.
View the system logbook for events and errors
As you prepare the instrument, you may run into an error that you want to
troubleshoot. You do this through the System Logbook Viewer.
•Click the Log icon ( ) in the toolbar of the Data Acquisition window, and
view the logged events.
•Or click Tools > System Logbook Viewer.
•Or right-click the icon in the system taskbar. First, click Enable
Notification. Then, right-click the LOG icon and click Configure. The system
can notify you of new errors and warning by showing messages from the
taskbar.
When the System Logbook Viewer is open, you can select the time period to keep
System Logbook entries. You can set the value from 1 week to 1 year. To do this,
you click Tools > Purge Settings. The System logbook purge dialog box opens.

Getting Started
Step 4. Set up and run an acquisition method
20 Agilent 6400 Series Triple Quadrupole LC/MS Quick Start Guide
Step 4. Set up and run an acquisition method
Read and follow the instructions in the online Help for each of the tasks
described on the following pages.
Also, do Exercise 1 of the Data Acquisition Familiarization Guide to learn how to
set up and run an acquisition method.
1Set up the method:
aType the values and settings for each of the tabs below.
bOptional. If you want to download the settings to the instrument, click
Apply.
cTo save the method, click Method > Save As.
dName the method and click OK.
2Enter values for all of the LC modules configured for the instrument.
NOTE Make sure when you type the MS parameters on the next page that the tune file is the one
that you want to use with the acquisition.