Agilent Technologies 6460 User Manual
Agilent MassHunter Workstation –
Data Acquisition for 6400 Series Triple
Quadrupole LC/MS
Familiarization Guide
For In Vitro Diagnostic Use
Before you begin 3
Prepare your system 3
Prepare to acquire data 4
Exercise 1 – Develop an acquisition method 6
Task 1. Enter acquisition parameters and acquire data 6
Task 2. Determine precursor ion masses 11
Task 3. Find optimum fragmentor voltage for maximum response 14
Task 4. Determine product ion masses 24
Task 5. Find optimum collision energy for MRM acquisition 30
Exercise 2 – Develop a Dynamic MRM method from an MRM acquisition
data file or an MRM method 33
Task 1. Create a batch file from an existing MRM data file 33
Task 2. Print a report in the Quantitative Analysis program 36
Task 3. Create a Dynamic MRM method using Update dMRM 38
Task 4. Create a Dynamic MRM method from an MRM method 40
Exercise 3 – Create a Triggered Dynamic MRM acquisition method 42
Task 1. Create a Triggered Dynamic MRM method from a Dynamic
MRM method manually 42
Task 2. Add/Modify compounds in an existing database 45
Task 3. Create a Triggered Dynamic MRM method from an existing
database 55
Exercise 4 – Optimize Acquisition parameters 59
Task 1. Use Optimizer to optimize acquisition parameters 59
2 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide
Use the exercises in this guide to learn how to use the Agilent 6460 Triple
Quad Mass Spectrometer (Model K6460). You can do these exercises with the
demo data files, SulfaDrugs, shipped with the system (in the Data folder of
your Qualitative Analysis installation disk), or with data you acquire.
In Exercise 1, you learn how to determine the best acquisition settings for
analyzing your compounds of interest. These instructions help you understand
not only how to set up a worklist to optimize instrument parameters for best
sensitivity in acquisition, but also how to use the Agilent MassHunter
Qualitative Analysis program to identify parameter values producing optimum
signal response. You can also learn about the Qualitative Analysis program by
using the Qualitative Analysis Familiarization Guide or the Qualitative
Analysis online Help.
In Exercise 2, you learn how to use either an acquired data file or the
Quantitative Analysis report results to update a Dynamic MRM method. This
method allows you to easily set up a Dynamic MRM method.
In Exercise 3, you learn how to create a triggered Dynamic MRM method.
In Exercise 4, you learn how to use two programs to optimize parameters.
Agilent MassHunter Optimizer helps you optimize acquisition parameters.
Specifically, it automates the selection of the best precursor ion and the
fragmentor voltage for the most abundant precursor ion, selection of the best
product ions, and optimization of collision energy values for each transition
for a list of compounds you specify.
Each task is presented in a table with three columns:
• Steps – Use these general instructions to proceed on your own to explore
the program.
• Detailed Instructions – Use these if you need help or prefer to use a
step-by-step learning process.
• Comments – Read these to learn tips and additional information about each
step in the exercise.
NOTE
See the Concepts Guide to learn more about how the triple quadrupole mass spectrometer
works and why the fragmentor and collision energy voltages are important. For
background information, see Chapter 3, “Triple Quadrupole MS and Sensitivity”, in the
Concepts Guide. See the online Help for detailed information on how the program works.
Before you begin
Prepare your system
Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 3
Before you begin
Before you begin, you need to check that your system is ready. If you plan to
acquire data, you also need to set up the instrument.
Prepare your system
1 Check that:
• The Data Acquisition program has been installed.
• The LC modules and the K6460 Mass Spectrometer have been configured.
• The performance has been verified.
• The system has been turned on.
If these actions have not yet been done, see the Installation Guide for your
instrument.
2 Copy the data files to your PC.
Copy the folder named SulfaDrugs in the Data folder on your Qualitative
Analysis installation disk to any location on your hard disk. This folder
contains all the data files needed for this exercise.
NOTE
Do not re-use the sulfa drug data files already on your system unless you know that you
copied them from the originals on the disk and you are the only one using them. Data files
that are already on the system may contain processed results, leading to different behavior
during the exercises in this guide.
Before you begin
Prepare to acquire data
4 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide
Prepare to acquire data
If you do not intend to acquire data but want to learn how to use the
Qualitative Analysis program for method development, you can skip this step,
which tells you how to prepare the demo sample. You then do those tasks that
show you how to use the Qualitative Analysis program with the sulfa drug
data files shipped with the system.
Parts List The exercise in this guide uses this equipment and materials:
• Agilent K1260 Infinity LC modules: well-plate sampler, binary pump,
thermostatted column compartment, DAD
• Zorbax column (see Table 1 on page 4)
• A 1 ng/μL concentration of the sulfa mix sample (prepared in this step)
1 Prepare the LC solvents.
For the A channel, add 1 mL of 5M ammonium formate to a 1-liter reservoir
filled with HPLC-grade water.
For the B channel, add 1 mL of 5M ammonium formate to a 1-liter reservoir
filled with 90:10 acetonitrile and HPLC-grade water.
2 Prepare the sample.
a Add 10 μL of the sulfa mix from one of the ampoules (500 μL) to 990 μL
of solvent A in a 2 mL glass sample vial so that the final concentration is
1 ng/μL.
b Cap the vial and place in a sample location in the autosampler.
3 Set up the LC column.
Use the column from Table 1. Other columns and instrument parameters
may be used in these exercises, but some parameters may need adjustment,
and the results will differ.
Table 1 Zorbax column
Triple Quadrupole Column Description Particle
Size
Pore Size Part
Number
6460 RRHD Eclipse Plus
C18.2.1 mm x 50 mm
1.8 µm 95Å 959757-902
Before you begin
Prepare to acquire data
Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 5
4 Set the column temperature to 60C. Lower temperatures may be used;
however, the retention times will be longer, and the pump pressure may
exceed the limit of some LC systems.
The Electrospray LC Demo Sample (P/N 59987-20033) contains five ampoules
with 100 ng/μL each of sulfamethizole (M+H)+ = 271, sulfamethazine (M+H)+ =
279, sulfachloropyridazine (M+H)+ = 285, and sulfadimethoxine (M+H)+ = 311.
Sulfamethizole Sulfamethazine Sulfachloropyridazine Sulfadimethoxine
NOTE
Determining optimal parameter values for acquiring sample compound data requires that
the Triple Quadrupole instrument already be tuned on the Tuning Mix calibrant ions. Before
proceeding with this exercise, make sure you have used Checktune or Autotune to verify
that calibrant ions each have the proper mass assignment, peak width, and signal
intensity.
See the Quick Start Guide, Installation Guide or online Help for instructions on tuning the
instrument.
Exercise 1 – Develop an acquisition method
Task 1. Enter acquisition parameters and acquire data
6 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide
Exercise 1 – Develop an acquisition method
For this exercise you analyze a mixture of four sulfonamide compounds. These
tasks show you how to manually select the acquisition parameters including
using the Qualitative Analysis program to analyze the data files. You can
instead use the automated process to select some of the acquisition
parameters. See “Exercise 4 – Optimize Acquisition parameters” on page 59 to
learn how to automate this process.
Task 1. Enter acquisition parameters and acquire data
In this exercise, you enter the conditions for the analysis of the sulfa drug mix.
l
Steps Detailed Instructions Comments
1 Enter LC parameters appropriate
for sulfa drug mix.
See Table 2.
a Double-click the Data Acquisition
icon.
b Make sure that Acquisition appears
as the selection in the Context text
box.
If Tune is the selection, click
Acquisition from the Context
dropdown menu in the Combo bar.
c Enter the LC parameters listed in the
Table 2.
• The Data Acquisition window
appears. See Figure 1 on page 8.
Table 2 LC parameters for sulfa drug mix
Parameter LC Parameter
PUMP
• Flowrate 800 µL/min
• Solvent A 5 mM ammonium formate in water
• Solvent B 5 mM ammonium formate in 90:10 acetonitrile:water
• Gradient (min - %B) 0 min - 13%
1.80 min - 60%
2 min - 60%
Exercise 1 – Develop an acquisition method
Task 1. Enter acquisition parameters and acquire data
Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 7
• Stop Time 2.5 min
• Post Time 3.0 min
INJECTOR
• Inj. Vol. 2.0 µL
• Injection Standard
• Draw Position 0.0 mm
UV DETECTOR
• Ch A 254 nm (4 nm BW on DAD)
• REF A (DAD only) 400 nm (80 nm BW)
COL THERM
• Temp 60 °C for the K6460 with an AJS ESI source
40 °C for ESI source
Table 2 LC parameters for sulfa drug mix (continued)
Parameter LC Parameter
Exercise 1 – Develop an acquisition method
Task 1. Enter acquisition parameters and acquire data
8 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide
Figure 1 MassHunter Workstation – Data Acquisition window
Steps Detailed Instructions Comments
2 Enter MS parameters appropriate
for sulfa drug mix and save the
method as iiiMS2Scantest.m,
where iii are your initials.
See Table 3 on page 9.
a Click the QQQ tab in the Method
Editor window.
b Select MS2 Scan from the Scan Type
list in the Time Segments table.
c Enter the other MS parameters as
listed in Table 3. These parameters are
in either the Acquisition or the Source
tabs.
d Save the method as
iiiMS2Scantest.m, where iii are your
initials.
Exercise 1 – Develop an acquisition method
Task 1. Enter acquisition parameters and acquire data
Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 9
Figure 2 Select Scan Type of MS2 Scan in the QQQ tab
Table 3 MS parameters for sulfa drug mix
Parameter Value (ESI) Value (AJS ESI)
• Inlet ESI (positive polarity) AJS ESI (positive polarity)
• Scan Type MS2 Scan MS2 Scan
• Delta EMV (+) 400 V 200 V
• Mass Range 100 to 400 100 to 400
• Cell Accelerator Voltage 7 V 7 V
• Gas Temp 350 °C 350 °C
• Gas Flow 12 L/min 10 L/min
• Nebulizer 50 psi 35 psi
• Sheath Gas Temperature not applicable 400 °C
• Sheath Gas Flow not applicable 12 L/min
• Nozzle Voltage not applicable 0 V
• Capillary Voltage positive 4000 V 4000 V
• Fragmentor 100 V 100 V
Exercise 1 – Develop an acquisition method
Task 1. Enter acquisition parameters and acquire data
10 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide
Steps Detailed Instructions Comments
3 Acquire data (optional).
• Set up a one-line worklist with
the method you just created.
• Name the data file
iiisulfamix01.d, where iii are
your initials.
• Designate a directory path to
hold your data files and method.
a If necessary, click View > Worklist to
display the Worklist window.
b Click Worklist > Worklist Run
Parameters. Verify that the
parameters are set properly. Click OK.
c Click Worklist > Add Multiple
Samples.
d Type iii
sulfamix01.d as the
data file name
e Select iiiMS2Scantest.m as the
method name.
f Click the Sample Position tab.
g Select the Autosampler, Well-plate or
Vial Tray.
h In the graphic, select a single position.
Click OK.
i In the Worklist window, mark the
check box to the left of the sample.
j Click the Start Worklist Run icon in
the main toolbar, the Run Worklist
icon in the Worklist toolbar or click the
Worklist > Run command.
• The Worklist window is tabbed with
the Method Editor window by
default. Click the Worklist tab to
show the Worklist window.
• The Number of samples is set to 1.
• You have just acquired a full scan
MS data file to see what ions are
being formed from the sample.
• This step is optional because you
can perform the next step with an
example data file that comes with
the program. If you prefer, you can
create your own data file as
described in this step.
Exercise 1 – Develop an acquisition method
Task 2. Determine precursor ion masses
Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 11
Task 2. Determine precursor ion masses
In this exercise, you determine the precursor ions for each of the sulfa drugs
in the acquired data file.
Steps Detailed Instructions Comments
1 Open the acquired data file.
• In the Qualitative Analysis
program, open either the
example file, sulfamix01.d, or
the data file you created in “Task
1. Enter acquisition parameters
and acquire data” on page 6.
a Double-click the Qualitative Analysis
icon.
The program displays the “Open Data
File” dialog box.
• When you open the sulfa drug
directory after installation, the Load
result data (lower left corner)
check box is grayed out.
• If you see the check box marked,
this means that the data file(s)
already contains results. Clear this
check box before opening the file.
Exercise 1 – Develop an acquisition method
Task 2. Determine precursor ion masses
12 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide
b Do one of the following:
• Select the example data file
sulfamix01.d, and click Open.
• Select the data file you created in
“Task 1. Enter acquisition
parameters and acquire data” on
page 6, and click Open.
By default, the system displays the
Total Ion Chromatogram (TIC).
• The figure below shows the default
layout.
• The Qualitative Analysis program
displays a newly opened data file
with the same layout and display
settings used for the previous data
file. Therefore, you MUST make
sure to return to the default
settings for this exercise.
Before you begin, make sure that all
previous settings are returned to their
default values:
• Restore default layouts
• Click Configuration > Window
Layouts > Restore Default
Layout.
• Make sure the method is default.m.
(see title bar)
• Click Method > Open.
• Select default.m, and click Open.
• Return display options to default
settings.
• In the Configuration menu, click
each of the Display Options
commands.
• Click Default, and then click OK.
Or...
• Restore the General layout.
• Click Configuration > Configure
for Workflow > General.
• Click OK.
• (optional) Save method changes
if needed.
• Return display options to default
settings.
• In the Configuration menu, click
each of the Display Options
commands.
• Click Default, and then OK.
Steps Detailed Instructions Comments
Exercise 1 – Develop an acquisition method
Task 2. Determine precursor ion masses
Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 13
2 Determine precursor ion masses
for all four peaks.
• You have determined them
correctly if you find the values
are similar to those shown in
this table:
• If you acquired the data file
using the Agilent Jet Stream
Technology, the retention times
may be different.
• The sulfamix01.d data file was
acquired with a different column
so your retention times are
different.
• Close the data file after finding
the precursor ion masses.
a In the Chromatogram Results window,
make sure that the Range Select icon
in the toolbar is on.
b Click the left mouse button and drag
the cursor across the first peak to
produce a shaded region, as in the
figure below.
c Right-click the shaded area, and click
Extract MS Spectrum from the
shortcut menu.
.
• The system displays an averaged
spectrum across the peak in the
MS Spectrum Results window.
• The precursor mass of the first
compound, sulfamethizole, is
determined to be m/z 270.9.
• To obtain a single scan, doubleclick the apex of the peak.
d Repeat step a through step c for the
other compounds.
The precursor ion masses should
match those in the table in step 2.
e Click File > Close Data File.
f When asked if you want to save the
results, click No.
• Some compounds form sodium
(Na) and/or potassium (K) adducts
as well, corresponding to M + 23
and M + 39 masses respectively.
Seeing these masses along with
the M + H can make for an easy
confirmation of which ion is the
pseudo-molecular ion (M + H)+.
Steps Detailed Instructions Comments
Exercise 1 – Develop an acquisition method
Task 3. Find optimum fragmentor voltage for maximum response
14 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide
Task 3. Find optimum fragmentor voltage for maximum response
Task 3 shows you how to carry out the optimization for fragmentor voltage by
creating selected ion-monitoring experiments for each compound within a
method and setting up multiple methods with varying fragmentor voltages.
Steps Detailed Instructions Comments
1 Set up six methods for six different
fragmentor voltages.
• Change to a SIM experiment.
• Use 60, 80, 100, 140, 180 and
220 volts as the fragmentor
voltages for the six methods.
• Save the methods as
iiiMS2SIMxxx.m, where iii are
your initials and xxx is the
voltage.
a In the Scan Type dropdown list, click
MS2 SIM.
Exercise 1 – Develop an acquisition method
Task 3. Find optimum fragmentor voltage for maximum response
Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 15
b In the Acquisition tab, enter the
Compound Name and Mass
(precursor ion mass) for
sulfadimethoxine (311 m/z).
c Right-click anywhere in the Scan
segments section, and click Add Row.
d Type the Compound Name and the
Mass for sulfachloropyridazine (285
m/z).
e Repeat steps c and d for
sulfamethazine (279 m/z) and
sulfamethizole (271 m/z).
f Save the method as iiiMS2SIM140.m,
where iii are your initials.
g Change the fragmentor voltage to 60,
and save the method as
iiiMS2SIM060, where iii are your
initials.
h Repeat step g for voltages 80, 100, 180
and 220, saving the methods as
iiiMS2SIM080, iiiMS2SIM100,
iiiMS2SIM180 and iiiMS2SIM220,
where iii are your initials.
• With the MS2SIM Scan Type set, a
different set of columns appears in
the Acquisition window.
• The Instrument Control and Data
Acquisition program creates a SIM
experiment for each compound
mass, starting with a default
fragmentor voltage of 140. See the
example below.
Steps Detailed Instructions Comments
Exercise 1 – Develop an acquisition method
Task 3. Find optimum fragmentor voltage for maximum response
16 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide
2 Set up and run the worklist
(optional).
• Set up six samples with Sample
Name SulfaDrugMix to inject 1ul
from vials 1-6 or the ones you
choose.
• Specify the data files as
iiiSulfaSIMxxx.d, where iii are
your initials and xxx is the
voltage.
a Click the Worklist icon if necessary to
make sure the worklist is visible.
b Click Worklist > New to start a new
worklist. You do not need to save the
last worklist.
c To set up the run, right-click the upper
left corner of the worklist, and click
Worklist Run Parameters.
d Type the paths for the method and
data files.
e Type the information for the 60 voltage
run.
f Click Worklist > Add Sample.
Another sample is added to the
Worklist. Add five samples to the
worklist for voltages 80-220.
g Mark the checkbox to the left of the
Sample Name for each of the six
samples.
• This step is optional because you
can use data files shipped with the
system to perform many of the
tasks in this exercise.
h Start the worklist.
• Click Worklist > Run.
• Click the icon in the main
toolbar.
• Click the icon in the worklist
toolbar.
• Note that the program only runs
those samples that are marked with
a checkmark.
• You can also run the worklist in
locked mode by clicking the lock
button in the main toolbar (the icon
looks like this icon when it is
locked).
Steps Detailed Instructions Comments
Exercise 1 – Develop an acquisition method
Task 3. Find optimum fragmentor voltage for maximum response
Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 17
3 Set up a qualitative method to
view the EIC data automatically.
• Open the data file
Sulfa_SIM60.d or your own
iiiSulfa_SIM60.d, where iii are
your initials.
• In the Method Editor, add in the
EICs corresponding to the
precursor ion masses of 271,
279, 285, and 311.
• Save the method as iiiExercise1,
where “iii” are your initials.
a Click File > Open Data File.
The system displays the Open Data
File dialog box
b Select either Sulfa_SIM60.d or
iiiSulfa_SIM60.d, and click Open.
c Click Method > Method Editor or
View > Method Editor.
The system displays the Method
Editor window.
• The Qualitative Analysis program
should be open. If not, see
“Double-click the Qualitative
Analysis icon.” on page 11.
• By default, the Method Editor is a
floating window. The window is
docked for these images. See the
online Help for more information on
floating and docking windows.
Steps Detailed Instructions Comments
Exercise 1 – Develop an acquisition method
Task 3. Find optimum fragmentor voltage for maximum response
18 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide
d If necessary, click Define
Chromatograms in the Chromatogram
section of the Method Explorer.
e To delete the BPC chromatogram, click
Delete in the Method Editor window.
f Select EIC for the Chromatogram
Definition Type,
g In the MS Chromatogram tab, make
sure MS Level is set to All and Scans
is set to All scan types.
h Clear the Do cycle sum check box.
i Type
271 as the m/z value.
j Click Add.
k Repeat steps i and j for the other
precursor ions,
279, 285 and
311.
l Click Method > Save As. The system
opens the Save As dialog box
m Save the method as iiiExercise 1.m.
n Click Save.
• The default Method Editor list
selection after installation is
Integrate (MS).
• You can also select Define
Chromatograms from the Method
Items list in the Method Editor
window.
• When you are defining
chromatograms, instead of deleting
the Defined chromatogram, you can
select EIC. Then, you enter the m/z
value and click the Change button.
Steps Detailed Instructions Comments
Exercise 1 – Develop an acquisition method
Task 3. Find optimum fragmentor voltage for maximum response
Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 19
4 Extract the chromatogram for the
data file and view the results.
• Make sure you can see all five
chromatograms, the TIC and
four EICs.
a Click the Run button on the Method
Editor toolbar to run the Extract
Defined Chromatogram command.
b To see the TIC and four EICs, click the
arrow next to the Maximum Number
of List Panes icon in the
Chromatogram Results toolbar, as
shown in the example below.
c Select 5 to view five chromatograms
simultaneously.
The system displays chromatogram
results as shown below.
• You can also click the
Chromatograms > Extract Defined
Chromatograms command to
extract the defined chromatograms.
Steps Detailed Instructions Comments
Exercise 1 – Develop an acquisition method
Task 3. Find optimum fragmentor voltage for maximum response
20 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide
5 Extract the remaining ion
chromatograms automatically.
• Extract Defined Chromatograms
should be the default action for
Assign File Open Actions.
• Open the remaining data files,
Sulfa_SIM80.d through
Sulfa_SIM220.d.
• Close the Method Explorer.
a Select File Open Actions from the
General section in the Method
Explorer.
b Make sure that Actions to be run list
only contains Extract Defined
Chromatograms.
c Click File > Open Data File.
The system displays the Open Data
File dialog box.
d Select the data files to be opened,
Sulfa_SIM80.d through
Sulfa_SIM220.d.
e Mark the Run ‘File Open’ actions
from selected method check box.
(lower left corner)
• The Qualitative Analysis Method
Editor lets you define actions to be
performed automatically upon
opening a data file(s).
Steps Detailed Instructions Comments
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