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 60C. 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

Exercise 1 – Develop an acquisition method

Task 3. Find optimum fragmentor voltage for maximum response

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 21

f Click Open.

The Qualitative Analysis program
displays all the EICs for all the data
files selected.

g To close the Method Explorer, Method

Editor and MS Spectrum Results
windows, click the X in the upper right
corner of each window.

• You can instead click View >
Method Explorer, View > Method
Editor, and View > MS Spectrum
Results.

Steps Detailed Instructions Comments

Mark this check box.

Exercise 1 – Develop an acquisition method

Task 3. Find optimum fragmentor voltage for maximum response

22 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

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 23

6 Select the fragmentor voltage that

produces the maximum response
for each of the precursor ions.

• Close the data files after you

determine the optimum voltage.

a In the Data Navigator window,

highlight the EICs for 271.0 m/z.

b Click the Show only the highlighted

items icon, .

Only the 271 m/z check boxes are
now marked.

c Look at the relative intensities of each

peak to determine which fragmentor
voltage setting will be best to use for
the 271 precursor.

d Repeat step a through step c for the

other three base peaks or precursor
ions.

e Click File > Close All.
f Click No in the Save dialog box.

• You press the Ctrl key to be able to

select multiple objects from the
Data Navigator window.

• You press the Shift key to be able to
select a group of objects.

• A fragmentor voltage of 100 should
be sufficient for each precursor ion.

• You can now determine the product
ions that are available for the
multiple-reaction monitoring
experiments to maximize sensitivity
for the analysis.

• You click Method > Save or

Method > Save As to save the

method changes.

• Click an EIC in the Data Navigator
window to change which chromatogram
is labeled in the Chromatogram Results
window. When the chromatogram label
color matches the color of the
chromatogram that has the highest
intensity, you use the Fragmentor
voltage that was used for that file.

Steps Detailed Instructions Comments

You can overlay the
chromatograms by
clicking the Overlaid
mode icon in the
Chromatogram Results
toolbar.

Exercise 1 – Develop an acquisition method

Task 4. Determine product ion masses

24 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

Task 4. Determine product ion masses

In this part of the method development, we will use three collision energies to
determine the best fragment ions to use for the eventual Multiple Reaction
Monitoring (MRMs) acquisition.

Steps Detailed Instructions Comments

1 Set up three product ion

acquisition methods and acquire
data.

• Use the MS parameters in the

example below, but change the
Fragmentor voltage to the
optimum voltage you determined
in the previous task.

• Save methods as iiiSulfamix

PI_xx.m, where iii are your

initials and xx is the collision
energy.

a Click the QQQ tab in the Method

Editor pane.

b Select

Product Ion in the Scan Type

combo box to scan each precursor ion
for all its product ions.

c Enter all MS parameters as listed in

the example below, making sure the
Collision Energy is set to

15 and the

Fragmentor voltage is set to the
optimum voltage determined in Task 3.

d Save the method as iiiSulfamix

PI_15.m.

e Repeat step c and step d for collision

energies of 30 and 45.

• When you change the Scan Type in
the Time Segments table, the Scan
segments table is reset. If you want
to copy the Scan segments to the
new Scan segments table,
highlight all of the lines in the Scan
segments table and then right-click
the Scan segments table and click
Copy. After you select a new Scan
Type, right-click the Scan segments
table and click Paste from

Clipboard.

• You cannot copy and paste the
Scan segments table between all
Scan Types.

2 Set up and run the worklist

(optional).

• Specify the data files as

iiiSulfamix PI_xx.d, where iii
are your initials and xx is the
collision energy.

a Click the Worklist tab.
b Add three samples to the worklist for

collision energies 15, 30 and 45.

c Mark the check box to the left of the

Sample Name for each sample you are
adding.

d Click Worklist > Run.

• This step is optional because you

can determine the product ion
masses from the data files shipped
with the system.

• Use the instructions in Step 2 of
Task 3 to set up the worklist.

Exercise 1 – Develop an acquisition method

Task 4. Determine product ion masses

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 25

3 Set up a qualitative method to

integrate and extract product ion
spectra.

• Use the data files

SulfamixPI_xx.d, where xx is

the collision energy, or your own
data files, iiiSulfamixPI_xx.d.

• Open Method Explorer and

Method Editor.

• Use TICs set up for MS/MS,

product ion and each of the
precursor ions 271, 279, 285,

311.

• Make sure the MS/MS

integrator has been selected and
the maximum number of peaks
has been limited to the largest
100 peaks.

• Add the ability to integrate and

extract peak spectra to the file
actions run upon data opening.

• Save the changes to the current

method.

a Click the Open Data File icon in the

toolbar.

b Select SulfamixPI_15.d.
c Clear the Run File Open Actions from

Specified Method check box, and
click Open.

d Make sure the Method Explorer and

the Method Editor windows are
displayed; otherwise, click the Method
Explorer and then Method Editor
icons.

e In the Chromatogram section in the

Method Explorer window, select

Define Chromatograms.

f Delete any existing chromatograms in

the Defined Chromatograms list.

g Select TIC from the Type list in the

Define chromatograms section.
h For MS level, select MS/MS.
i Mark the Do cycle sum check box.
j For Scans, select Product ion.
k For Precursor ion m/z, type

271.

l Click the Add button.
m Repeat steps j and k for each ion.

• The Qualitative Analysis program

should already be open and contain
iiiexercise 1.m as the method.

Steps Detailed Instructions Comments

Exercise 1 – Develop an acquisition method

Task 4. Determine product ion masses

26 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

n From the Method Explorer in the

Chromatogram section, click

Integrate (MS/MS).
o Select MS/MS as the Integrator

selection, if necessary.

• These data files contain MS/MS

data, so you need to modify the
parameters in the Integrate
(MS/MS) section. If the data file
contained only MS data, you would
need to modify the parameters in
the Integrate (MS) section.

Figure 3 Integrate (MS/MS) > Integrator Tab

p Click the Peak Filters tab. Make sure

that the Limit (by height) to the

largest check box is marked and set to

the value

100 as shown below.

Figure 4 Integrate (MS/MS) > Peak Filters tab

Steps Detailed Instructions Comments

Exercise 1 – Develop an acquisition method

Task 4. Determine product ion masses

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 27

q Click General in Method Explorer, and

then click File Open Actions.
r Select Integrate and extract peak

spectra from the Available actions list

and click to add this to

Actions to be run.

Figure 5 General > File Open Actions tab

s To apply the changes to the current

method, iiiexercise1.m, click the Save

Method icon. You can also click

Method > Save.

4 Run the qualitative method on the

current data file.

• In the Method Editor toolbar, click the

Run button, . When the Assign

Actions to Run Opening A Data File
section is displayed, the Actions to be

run list is run.

• The program first extracts the

product ion chromatograms for
each precursor ion in the data file.

• Next, it finds the largest peak in the
total ion chromatograms, and
integrates and extracts peak
spectra from each integrated peak.

• See Figure 6 on page 28.

Steps Detailed Instructions Comments

Exercise 1 – Develop an acquisition method

Task 4. Determine product ion masses

28 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

Figure 6 Results for integration and extraction of peak spectra.

5 Run the ‘File Open’ actions on the

remaining product ion data files.

• Use either the example files,

Sulfamix PI_xx.d, or the data

files you acquired in step 2.

a Click File > Open Data File.

The system displays the Open Data
File dialog box.

b Hold the Ctrl key and do one of these:

• Select the two data files Sulfamix

PI_30.d, and Sulfamix PI_45.d.

• Select the data files you acquired in

step 2.

c Mark the Run ‘File Open’ actions

from selected method check box in

the Open Data File dialog box, and
click Open.

• After the data files open, the
Qualitative Analysis method first
extracts the product ion
chromatograms for each precursor
ion.

• Next, it integrates each total ion
chromatogram and extracts peak
spectra from each integrated peak.

Steps Detailed Instructions Comments

Exercise 1 – Develop an acquisition method

Task 4. Determine product ion masses

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 29

6 Identify product ions.

• View each set of TICs and

spectra individually (e.g., 271
m/z first).

• Close the data files.

a In the Data Navigator, select the TICs

and spectra for the 271 m/z precursor
ion.

b Click the Show only the highlighted

items icon, .
c Click View > MS Spectrum Peak List 1.
d Examine the spectra to see which

fragment ions are produced at which

collision energies.
e Repeat steps a to d until all the

product ions are identified.

f Click the Close Data File icon in the

main toolbar, and click Close when the

dialog box containing the list of data

files pops up.

• The m/z 155.7 product ion is the
most abundant of any product ion
and the highest signal is recorded
at 15 V. This means that a good
choice for the MRM for
sulfamethizole would be 271.0 >

155.7 when the collision energy is
around 15 V.

• The peak may not be labeled if the
peak is too wide.

•

• The product ions appear to be:

Sulfamethizole-271.0 > 155.7
Sulfamethazine-279.0 > 185.7
Sulfachloropyridazine-285.0 > 155.7
Sulfadimethoxine-311.0 > 155.7

Steps Detailed Instructions Comments

Exercise 1 – Develop an acquisition method

Task 5. Find optimum collision energy for MRM acquisition

30 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

Task 5. Find optimum collision energy for MRM acquisition

In this task, you set up MRM acquisition methods for the sulfa drugs for
different collision energies. By examining the spectra and comparing peak
intensities, you determine the optimal collision energy settings for the
compounds.

Steps Detailed Instructions Comments

1 Set up three MRM acquisition

methods.

• Use all the MS parameters in the

example below except for the
collision energy value.

• Use collision energies of 10, 15

and 20.

• Save methods as iiiSulfamix

MRM_xx.m, where iii are your

initials and xx is the collision
energy.

a Click the QQQ tab.
b Set Scan Type to MRM.
c Enter all MS parameters shown in the

example below except for the collision
energy value.

d In the collision energy column, type

10 for each compound.

e Save the method as iiiSulfamix

MRM_10.m.

f Repeat step d and step e for collision

energies of 15, 20, 25, 30 and 35
saving the methods as iiiSulfamix
MRM_xx.m, where iii are your initials
and xx is the collision energy.

• Because the largest peaks were
produced with a collision energy of
15 in the previous exercise, you will
look at only those collision energies
to either side of 15.

Exercise 1 – Develop an acquisition method

Task 5. Find optimum collision energy for MRM acquisition

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 31

2 Set up and run the worklist

(optional).

• Specify the data files as

iiiSulfamix MRM_xx.d, where
iii are your initials and xx is the

collision energy.

a Click the Worklist tab to make the

worklist visible.

b Add six samples to the worklist for

collision energies 10, 15, 20, 25, 30, 35.

c Mark the check box to the left of the

Sample Name for each of the three
samples.

d Click Worklist > Run.

• This step is optional because you

can use the six example data files
in the next step.

3 Compare the compound transition

intensities at different collision
energies.

• Open the MRM data files:

SulfamixMRM_10.d
SulfamixMRM_15.d
SulfamixMRM_20.d
SulfamixMRM_25.d
SulfamixMRM_30.d
SulfamixMRM_35.d

• Set the MRM chromatogram

extraction parameters as shown
at right for all transitions.

• Disable the TICs for clarity and

examine the peak intensities.

• Compare the intensities of each

compound transition obtained at
one collision energy with the
same compound transition
obtained at another collision
energy. (Do this in Overlaid
Mode with all the MRM
chromatograms.)

• Close the data files but don’t

save results.

• Refer to Table 4 on page 32 for

optimal method settings for
each compound.

a Open the Qualitative Analysis

program.

b Clear the Run ‘File Open’ actions...

check box.

c Open the MRM data files in the

Qualitative Analysis program.

d Right-click the Chromatogram Results

window, and click Extract
Chromatograms from the shortcut
menu.

e To select all data files, click the last

file while holding down the Shift key.

f Enter the parameters as listed in the

example below, and click OK.

g Clear the TIC check boxes to make the

MRM chromatograms easier to view.

• Why a spectrum for MRM? It’s a
feature of the program to show
spectra even for MRM experiments
and can be quite handy for
comparing relative intensities of
product ions generated from the
same precursor.

• You can also click Chromatograms
> Extract Chromatograms to start

this dialog box.

Steps Detailed Instructions Comments

Exercise 1 – Develop an acquisition method

Task 5. Find optimum collision energy for MRM acquisition

32 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

h Click the Overlaid Mode icon, .
i Compare peak intensities for each

compound transition in each data file
in the Chromatogram Results window.

• Compare the colors shown in
Chromatogram Results with the
color next to the MRM transition
name in the Data Navigator.

• You can also right-click the
Chromatogram Results window
header and compare the colors of
the chromatograms to the colors of
the titles in the shortcut menu.

Unless you decide to acquire MRMs at
lower collision energies, you should
find that the optimal method settings
are as shown in Table 4.

j Click the Close Data File icon in the

main toolbar, and click Close when the
Close Data File dialog box appears.

• You now have all the information
you need to do an MRM acquisition
experiment of the sulfa drug
mixture. Consider doing at least
one more run with those settings.

Steps Detailed Instructions Comments

Table 4 Compounds and Collision Energy

Compounds MRM Transition Collision Energy (V)

Sulfamethizole 271.0 > 155.8 10

Sulfamethazine 279.0 > 185.7 15

Sulfachloropyridazine 285.0 > 155.7 10

Sulfadimethoxine 311.0 > 155.7 15

Exercise 2 – Develop a Dynamic MRM method from an MRM acquisition data file or an MRM method

Task 1. Create a batch file from an existing MRM data file

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 33

Exercise 2 – Develop a Dynamic MRM method from an MRM
acquisition data file or an MRM method

The purpose of this exercise is to create a Dynamic MRM method from an
acquired MRM data file for sulfamix_MRM data files with the correct retention
times for Dynamic MRM using the Quantitative Analysis program.

For this exercise, you have three main tasks:

• “Task 1. Create a batch file from an existing MRM data file” on page 33

• “Task 2. Print a report in the Quantitative Analysis program” on page 36

• “Task 3. Create a Dynamic MRM method using Update dMRM” on page 38

You can easily create a Dynamic MRM method from an existing MRM method.

• “Task 4. Create a Dynamic MRM method from an MRM method” on page 40

Task 1. Create a batch file from an existing MRM data file

In this exercise, you create a batch and a method from an existing MRM data
file.

Steps Detailed Instructions Comments

1 Open the Quantitative Analysis

program and create a batch file
with one sample file,
SulfamixMRM_10.d.

• Copy the data file

SulfamixMRM_10.d from the
installation disk to the

\MassHunter\Data\MRM_to_
DMRM folder.

a Double-click the QQQ Quantitative

Analysis icon or the Drug Quant

(QQQ) icon.
b Click File > New Batch.
c Navigate to the \MassHunter\Data\

MRM_to_DMRM folder.
d Type

MRM_to_DMRM in the File

name text box.
e Click Open.
f If the Add Samples dialog box does

not open, click File > Add Samples.
g Select the file SulfamixMRM_10.d.
h Click OK.

• The file SulfamixMRM_10.d is on

the installation disk in the
\Support\Data folder. Copy this
entire folder to the

\MassHunter\Data\
MRM_to_DMRM folder.

Exercise 2 – Develop a Dynamic MRM method from an MRM acquisition data file or an MRM method

Task 1. Create a batch file from an existing MRM data file

34 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

2 Create a method for that batch

using MRM data.

a Click Method > New > New Method

from Acquired MRM Data.
b Select the SulfamixMRM_10.d data

file.

c Click Open.

3 Set the Concentration Setup,

Qualifier Setup, and Calibration
Curve Setup.

• Add calibration level 1 with a

concentration of 10000.

• Set the Uncertainty to Relative

for all qualifiers.

• Set the Curve Fit to Linear.

• Set the Curve Fit Origin to

Include.

• Set the Curve Fit Weight to

None.

a Select Concentration Setup in the

Manual Setup Tasks section in the
Method Tasks pane.

b Select the first compound in the table.
c Right-click the compound row and

click New Calibration Level from the
shortcut menu.

d Enter

A1 in the Level column and 10

in the Conc. column.

e Right-click in the Level box and click

Copy Calibration Levels To.
f Click Select All. Click OK.
g Select Qualifier Setup in the Manual

Setup Tasks section in the Method

Tasks pane.

h Verify that the Uncertainty is Relative.
i Select Calibration Curve Setup in the

Manual Setup Tasks section in the

Method Tasks pane.
j Set Curve Fit to Linear for all

compounds.
k Set CF Origin to Include for all

compounds.
l Set CF Weight to None for all

compounds.

• Refer to the online Help in the
Quantitative Analysis program for
additional help on these tasks.

• You can also click Method > Copy
Calibration Levels To to display the
Copy Calibration Levels To dialog

box.

• To enter the same value in all cells
in a column, you can change the
value in the first row, and then
right-click that value in the first row
and click Fill Down.

• The compound names in
Quantitative Analysis need to
exactly match the Compound Name
in the QQQ Acquisition program. If
you capitalized the Compound
Name in the Data Acquisition
program, then you need to make
sure that the Name in the
Quantitative Analysis program is
also capitalized.

Steps Detailed Instructions Comments

Exercise 2 – Develop a Dynamic MRM method from an MRM acquisition data file or an MRM method

Task 1. Create a batch file from an existing MRM data file

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 35

•

4 Verify method and then save the

method and apply the method to
the batch.

a Click Method > Validate.
b Click OK on the message box. Fix any

errors, if necessary.

c Click Method > Save As.
d Enter

MRM_to_DMRM.

e Click the Save button.
f Click Method > Exit.
g (optional) Click Analyze in the Apply

Method dialog box.

h Click Yes to apply the method to the

batch.

• In the Apply Method dialog box,
you can click Analyze to
automatically start additional batch
processing.

5 Analyze and save the batch. a Click Analyze > Analyze Batch.

b Click File > Save Batch.

Steps Detailed Instructions Comments

Exercise 2 – Develop a Dynamic MRM method from an MRM acquisition data file or an MRM method

Task 2. Print a report in the Quantitative Analysis program

36 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

Task 2. Print a report in the Quantitative Analysis program

In this task, you print a report using any template.

You can update a Dynamic MRM method using either a data file or a
quantitation report folder, so this task creates the quantitation report folder.

Steps Detailed Instructions Comments

1 Print a report using the template

MRM_to_DMRM.xltx.

a Click File > Save Batch.
b Click Report > Generate.

The system displays the Generate
Report dialog box.

c Select the Report folder. This folder

name will be used in the next task.

d Select the Report method.
e Click Edit. The Report Method Edit

dialog box opens.

f Click the Results tab. Click Yes

Always generate results file.
g Click Save & Exit.
h Mark All samples.
i Mark All compounds.
j Click OK.

• Copy the MRM_to_DMRM.xltx

template from the \Support\Data
folder on the installation disk.

• For this report, you do not need to
print the report.

• If you have not created a Report
Method, see the online Help for
Quantitative Analysis for
instructions on how to create a
report method.

• The Data Acquisition program uses
the results file in the Quant reports
folder to update the method, so you
must generate this file if you want
to update your method with it.

2 Check the status of the report

using the Queue Viewer program.

a Click Report > Queue Viewer.
b Wait for the report to finish printing.
c Close the Task Queue Viewer

program.

Exercise 2 – Develop a Dynamic MRM method from an MRM acquisition data file or an MRM method

Task 2. Print a report in the Quantitative Analysis program

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 37

Steps Detailed Instructions Comments

Exercise 2 – Develop a Dynamic MRM method from an MRM acquisition data file or an MRM method

Task 3. Create a Dynamic MRM method using Update dMRM

38 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

Task 3. Create a Dynamic MRM method using Update dMRM

You can create a Dynamic MRM method from an MRM data file or a
Quantitative Analysis method. You first set the Scan Type to Dynamic MRM,
and then you use the Update MRM Method dialog box.

Steps Detailed Instructions Comments

1 Open the method iiiSulfamix

MRM_10.m and save it to a new
name with the format iiiSulfamix
dMRM.m, where iii are your
initials.

a Click File > Open > Method.
b Select the iiiSulfamix MRM_10.m

method. Click OK.
c Click Method > Save As.
d Type the new method name with the

format iiiSulfamix_dMRM.m.

• In this example, the batch is in the

\MassHunter\Data\
MRM_to_DMRM folder.

2 Change the method to a dynamic

MRM method with the same
compounds. You can either use a
data file or the report that was
generated in the last task.

a Click the Acquisition tab in the QQQ

tab in the Method Editor window.
b Right-click the Scan segments table

and click Update DMRM Method. The

Dynamic MRM Update Options dialog

box opens.
c Select the folder containing the

report.results.xml file or the data file

iiiSulfamix MRM_10.d.

d Mark Add new compound/transition.
e Mark Update retention time.
f Mark Update trigger window.
g Click OK.

• The Dynamic MRM Update tool
automatically sets the Scan type to
Dynamic MRM.

• You can select either a data file that
was acquired with a Scan Type of
MRM or a Quant Report folder as

the input to this dialog box. The
Scan segments are created from
one of these two input sources.

You can update the compounds in the Scan
segments table by using a QQQ data file or a
Quantitative analysis report folder.

If you select a Quantitative analysis report
folder, you need to make sure to generate the
results file. See “Task 2. Print a report in the

Quantitative Analysis program” on page 36 to

learn how to always generate the results file.

Exercise 2 – Develop a Dynamic MRM method from an MRM acquisition data file or an MRM method

Task 3. Create a Dynamic MRM method using Update dMRM

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 39

•

h Verify that each row has a Compound

Name. A blank Compound Name is

not allowed.

i Click Method > Save.

Steps Detailed Instructions Comments

The compounds from the data file or quantitation report are automatically added to the Scan segments table.

Exercise 2 – Develop a Dynamic MRM method from an MRM acquisition data file or an MRM method

Task 4. Create a Dynamic MRM method from an MRM method

40 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

Task 4. Create a Dynamic MRM method from an MRM method

You can create a Dynamic MRM method directly from an MRM method by
using the Paste from Clipboard command from the shortcut menu. You need
to manually enter the retention time.

Steps Detailed Instructions Comments

1 Open the method iiiSulfamix

MRM_10.m and save it to a new
name with the format iiiSulfamix
dMRM2.m, where iii are your
initials.

a Click File > Open > Method.
b Select the iiiSulfamix MRM_10.m

method.

c Click OK.
d Click Method > Save As.
e Type the new method name with the

format iiiSulfamix_dMRM2.m.

f Click the Save button.

2 Copy all compounds from the Scan

segments table in the MRM
method.

a Click the Acquisition tab in the QQQ

tab in the Method Editor.

b Select all of the rows in the Scan

segments table.

c Right-click the Scan segments table

and click Copy.

• To select all of the rows in the Scan
segments table, you select the first
row in the table, Then, you scroll to
the last row in the Scan segments
table. Press the Shift key and select
the last row in the table.

3 Change the Scan Type to Dynamic

MRM and paste the rows into the
new Scan segments table.

a Select Dynamic MRM for the Scan

Type.

b Right-click the Scan segments table

and click Paste from Clipboard.

c Click Method > Save.

• To combine multiple Time

Segments into one Dynamic MRM
Time Segment, you paste the Scan
segments into Excel and create one
long list. Then, you copy all of the
Scan segments in Excel.

4 Enter the retention times and

delete the original compound.

a Type the value for the Ret. Time (min)

for Sulfachloropyridazine.

b Type the value for the Ret. Time (min)

for Sulfadimethoxine.

c Type the value for the Ret. Time (min)

for Sulfamethazine.

d Type the value for the Ret. Time (min)

for Sulfamethizole.

e Select the original compound in the

Scan segments table.

f Right-click and click Delete Row.
g Click Method > Save.

• Retention time is different for

different systems and columns. For
the example data files, enter the
following values:

• Sulfachloropyridazine: 0.65

minutes

• Sulfadimethoxine: 2.03 minutes

• Sulfamethazine: 0.98 minutes

• Sulfamethizole: 0.37 minutes

Exercise 2 – Develop a Dynamic MRM method from an MRM acquisition data file or an MRM method

Task 4. Create a Dynamic MRM method from an MRM method

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 41

•

Steps Detailed Instructions Comments

Exercise 3 – Create a Triggered Dynamic MRM acquisition method

Task 1. Create a Triggered Dynamic MRM method from a Dynamic MRM method manually

42 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

Exercise 3 – Create a Triggered Dynamic MRM acquisition
method

For this exercise you analyze a mixture of four sulfonamide compounds.

Task 1. Create a Triggered Dynamic MRM method from a Dynamic
MRM method manually

You can create a Triggered Dynamic MRM method directly from a Dynamic
MRM method. In a Triggered Dynamic MRM method, you specify some of the
transitions to be primary transitions. These transitions are acquired for the
entire retention time window. Some of these primary transitions are also
marked as triggers. As the data is acquired, the program checks whether or
not the abundances of the trigger transitions are higher than the threshold. If
the abundances are higher than the thresholds and other additional
conditions are met, then the secondary transitions are acquired. These other
conditions are described in the Concepts Guide.

Steps Detailed Instructions Comments

1 Open the method iiiSulfamix

dMRM2.m, where iii are your

initials.

a Click File > Open > Method.
b Select the iiiSulfamix_dMRM2.m

method.

c Click OK.
d Click Method > Save As.
e Type the new method name with the

format

iiiSulfamix_TriggeredDMRM.m.

f Click the Save button.

• A Triggered Dynamic MRM method

is a type of Dynamic MRM method.
The Scan Type for both methods is

Dynamic MRM.

• The Dynamic MRM method is the

template method for the
optimization.

Exercise 3 – Create a Triggered Dynamic MRM acquisition method

Task 1. Create a Triggered Dynamic MRM method from a Dynamic MRM method manually

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 43

2 Change the method to a triggered

dynamic MRM method.

a Click the Acquisition tab in the QQQ

tab in the Method Editor.

b Mark the Triggered check box in the

Triggered MRM section. This section
is only available if the Scan Type is

Dynamic MRM.

c Select whether to automatically mark

the highest product ion as the Primary.

d Enter the value for Repeats.

• Several columns are added to the
Scan segments table. These

columns only apply to a triggered
dynamic MRM method.

• The value Repeats is the number of
times to acquire each of the
secondary transitions when the
triggering conditions are met.

3 Add additional transitions. See the

next image.

a Select the first compound. Right-click

and click Insert Row. Repeat to insert
another row

b Modify the information in these rows

to match the information in the next
image.

c Repeat for each compound.

• For each compound, we are going

to add additional transitions.

4 Select the transitions that are the

Primary transitions. See the next

image.

a For each transition, mark the Primary

check box if it is a Primary transition.

b Verify that you have marked at least

one transition as the Primary
transition for each Compound Name.

• You can select multiple transitions
from each compound to be Primary
transitions. If a transition has the
same Compound Name, then it is
part of the same compound. You
must mark at least one transition as
a Primary transition for each
compound.

• Typically, the most abundant ion is
the Trigger transition.

Steps Detailed Instructions Comments

Exercise 3 – Create a Triggered Dynamic MRM acquisition method

Task 1. Create a Triggered Dynamic MRM method from a Dynamic MRM method manually

44 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

5 Select the transitions that are the

Trigger transitions and set the

trigger conditions.

a For each compound, mark the Trigger

check box if it is a Trigger transition.

b (optional) Mark a second Trigger

transition.

c Enter the Threshold value for each

Trigger transition.

d Enter the Trigger Entrance for each

Trigger transition.

e Enter the Trigger Delay for each

Trigger transition.

f Enter the Trigger Window for each

Trigger transition.

• For each compound, you can have

two Trigger transitions.

• If the Trigger transition has an
abundance over the Threshold,
then that triggering condition is
met.

• By default, the Trigger Entrance,
the Trigger Delay and the Trigger
Window are set to 0. If these
values are 0, then these triggering
conditions are not enabled.

• The threshold is established when
the method is updated using a data
file. The other values are also
selected based on results. You must
collect data at different settings to
establish what works best. These
values are very important to the
success of the method.

•

Steps Detailed Instructions Comments

Exercise 3 – Create a Triggered Dynamic MRM acquisition method

Task 2. Add/Modify compounds in an existing database

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 45

Task 2. Add/Modify compounds in an existing database

You can also manually add compounds to a database and modify the
compounds in the database. In the next task, you create a Triggered Dynamic
MRM method from the compounds in the database.

Steps Detailed Instructions Comments

1 Review the

iiiSulfamix_dMRM2.m, where iii
are your initials.

a Click File > Open > Method.
b Select the iiiSulfamix_dMRM2.m

method.

c Click OK.
d Review the parameters.

• A Triggered Dynamic MRM method

is a type of Dynamic MRM method.
The Scan Type for both methods is

Dynamic MRM.

2 Start the MassHunter Optimizer

program.

• Double-click the Optimizer icon. . • If you are optimizing peptides, use
the Optimizer for Peptides program.

3 Set parameters on the Optimizer

Setup tab.

a Click the Optimizer Setup tab.
b Click the Injection (with or without

column) button.

c Set the CE range from

4 to 48.

d Set the Cell Accelerator Voltage to

7.

e Right-click the table and click Add

Method.

f Select the iiiSulfamix_dMRM2.m

method.

• To create low mass product ions
from a precursor ion near 300 m/z,
you need fairly high collision
energies.

4 Set parameters on the Precursor

Ion Selection tab.

a Click the Precursor Ion Selection tab.
b Verify that +H is marked for the

Positive ions (with priorities) list.

5 Set parameters on the Product Ion

Selection tab.

a Click the Product Ion Selection tab.
b Click the Mass (m/z) button under

Low mass cut-off.

c Enter

60 for the low mass cut-off.

• On the Product Ion Selection, you
can automatically add up to 4
product ions per compound (for
instance, 2 primaries and 2
secondaries). You want 8 to 10
peaks in the composite spectrum to
prove that this is indicative of the
compound, so you need to add at
least some of the product ions
manually.

Exercise 3 – Create a Triggered Dynamic MRM acquisition method

Task 2. Add/Modify compounds in an existing database

46 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

6 Set parameters on the Compound

Setup tab and add additional
transitions.

• For Precursor ion 311, add the

following product ions: 245.1,

218.1, 172.1, 140.3, 107.1

• For Precursor 285, add the

following product ions: 108.1,

92.1, 80.1, 65.1, 39.2

• For Precursor 279, add the

following product ions: 185.7,

155.6, 107.7, 92.1

• For Precursor 271, add the

following product ions: 177.8,
115, 91.3, 80.1, 64.8

a Click the Compound Setup tab.
b Click the Import/Export > Import

from Acquisition Methods command.

c Select the iiiSulfamix_dMRM2.m

method and click Open.

d (optional) Right-click the tab and click

Expand/Collapse All Rows.

e Select one of the Product rows for

one of the compounds. In this
example, select the Product row 155.7
for Precursor 311.

f Right-click the Product row and click

Add Product Ion. In this example, you

add 5 product ion rows.

g Enter the Product in each of the

product ion rows that were added. See

“To determine product ions in the
Qualitative Analysis program:” on

page 47.

h Add product ions for the other three

compounds.

• For each compound, we are going
to add additional transitions.

• In the Qualitative Analysis program,
you examine Product Ion data files
which you acquired previously to
determine additional transitions to
add. See “Task 4. Determine

product ion masses” on page 24.

• You can use the arrow keys to move
between rows in the Product table.

• Some of the product ions were
determined from the Veterinarian
Drugs tMRM database.

• See Table 5 on page 54 for values to
use in the method.

Steps Detailed Instructions Comments

This product ion scan has a
precursor mass of 311. You
examine the MS spectrum to
determine the product ions to
add to the Product ion section
of the Compound Setup table.

The product ions that are manually added as
additional Product ions in Optimizer are shown in
the MS Spectrum Results window. The green
boxes were added in this guide to show which
product ions were used.

Exercise 3 – Create a Triggered Dynamic MRM acquisition method

Task 2. Add/Modify compounds in an existing database

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 47

• To determine product ions in the

Qualitative Analysis program:

a Open the SulfamixPI_15.d from “Task

4. Determine product ion masses” on

page 24.

b Click Find > Find Compounds by

Targeted MS/MS.
c Close the Compound List window.
d Select a compound in the Data

Navigator window. For this example,

click Cpd 4.
e Click the Autoscale Y-axis icon in the

MS Spectrum Results toolbar.
f Right-click and drag to zoom in on the

MS spectrum.

• If possible, rearrange the windows
on the screen so you can see the
Optimizer program and the
Qualitative Analysis program at the
same time.

• See Table 5 on page 54 for values to
use in the method.

7 Set other parameters in the

Compound Setup tab and start the
optimization.

• You cannot perform a

multi-compound run.

• You have to mark each row in

the table to use.

a Mark the check box in the left column

at the top of the table. The check box
for every row in the table is marked.

b Clear the Perform multi-compound

run check box in the right column.

c Click the Start Optimization button in

the Optimizer toolbar.

• You cannot perform a
multi-compound run with the
number of transitions that were
added. If you mark this check box,
then the Expected peak width
(base) is automatically set to
almost 80 seconds wide. If you
clear this check box, then the
Expected peak width is calculated
to be around 9 seconds which is
more appropriate.

• See Table 5 on page 54 for values to
use in the method.

Steps Detailed Instructions Comments

This product ion scan has a
precursor mass of 311. You
examine the MS spectrum to
determine the product ions
to add to the Product ion
section of the Compound
Setup table.

Exercise 3 – Create a Triggered Dynamic MRM acquisition method

Task 2. Add/Modify compounds in an existing database

48 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

8 Examine the Optimizer Report. a Examine the Collision Energy for each

Product Ion.

b Print or save the report.

9 Save the compounds. • Click the File > Save Compounds

command.

10 Import compounds from a

database.

• Click the Import/Export > Import from
Database command. The Database

Browser program opens.

• You can also import compounds
that were distributed as part of a
database.

Steps Detailed Instructions Comments

As a general rule, as the Product Ions get smaller,
the optimal Collision Energy gets larger. However,
when you also examine the abundance, you can
see that if the Collision Energy is set to 48 for the
smallest product ion, the smallest product ion can
become the dominant peak. The collision energies
are further adjusted later in this task.

This report was generated with a different set of
transitions.

Exercise 3 – Create a Triggered Dynamic MRM acquisition method

Task 2. Add/Modify compounds in an existing database

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 49

11 In the Database Browser program,

select the transitions.

a Mark the Show All Records check

box.

b Click the Select top button under

Select Transitions.

c Type

10 for the ranked transitions.

d Click the Select Transitions button.

• All the transitions that you typed in

are visible.

• The tools to allow you to set up
Primary transitions and Secondary
transitions are available in this

program.

• If any of the compounds have two
collision energies, clear the check
boxes for one of these values.

• See Table 5 on page 54 for values to
use in the method.

Steps Detailed Instructions Comments

Exercise 3 – Create a Triggered Dynamic MRM acquisition method

Task 2. Add/Modify compounds in an existing database

50 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

12 In the Database Browser program,

automatically select the Primary
transitions and Trigger transition.

a In the Set top ranked transitions as

primary box, enter 2.

b Click the Set Primaries and Trigger

button.

• The program automatically selects
the two most abundant transitions
as the Primary transitions.

• The program also selects the most
abundant transition as the Trigger.

• You can manually select a second
Trigger transition.

• See Table 5 on page 54 for values to

use in the method.

Steps Detailed Instructions Comments

You examine the Primary column and the Trigger column to determine which transitions are selected. You can select
one or two Trigger transitions. You can select multiple Primary transitions.

Exercise 3 – Create a Triggered Dynamic MRM acquisition method

Task 2. Add/Modify compounds in an existing database

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 51

13 Review the Primary transitions and

Trigger transitions.

• For sulfachloropyridazine, select

285 m/z -> 155.7 m/z transition
as the Primary and Trigger
transition.

• For sulfadimethoxine, select 311

m/z -> 155.7 m/z transition as
the Primary and Trigger
transition.

• For sulfamethazine, select 279

m/z -> 185.7 m/z transition as
the Primary and Trigger
transition.

• For sulfamethizole, select 271

m/z -> 155.8 m/z transition as
the Primary and Trigger
transition.

• Review each compound. Change the
Primary and Trigger transitions to the
transitions listed in the left column.

• Change the other Primary transitions
as shown below.

• The program selected the most
abundant transitions which in this
example often had a low m/z for
the Product Ion. A very abundant
low m/z ion may be unsuitable as a
Primary transition.

• You can select two Primary
transitions as triggers for a
compound.

• In the example below, all of the
columns have values. The
Fragmentor voltages and collision
energy values were set in
Optimizer.

• See Table 5 on page 54 for values to
use in the method.

Steps Detailed Instructions Comments

Exercise 3 – Create a Triggered Dynamic MRM acquisition method

Task 2. Add/Modify compounds in an existing database

52 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

14 Review the Import List table on

the Import List tab.

a Click the Add to Import List button.
b Click the Import List tab.
c Review the Import List table.

• In this example, you are importing

from the database to the Import
List. Then, you are importing from
Database Browser to Optimizer.

15 Review the Compound Setup table

in Optimizer. You replace all
compounds with the compounds
from the Database Browser
program.

a Click the Import button.
b Click the Yes to All button.
c In the Compound Setup tab in

Optimizer, review the compounds.

• The compounds in Optimizer are
overwritten by the compounds that
you updated in the Database
Browser program.

Steps Detailed Instructions Comments

Exercise 3 – Create a Triggered Dynamic MRM acquisition method

Task 2. Add/Modify compounds in an existing database

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 53

16 Save the new compound

parameters to the database.

• Click the File > Save Compounds
command to save all of the changes to
the database.

• You cannot see these results by
default, but the Primary and Trigger
transitions are updated in the
project.

• The Primary column, Trigger
column, Trigger Entrance Delay
column, Trigger Delay column,

Trigger Window column and
Trigger MRM Threshold column

are available in the Compound
Setup tab. They may be hidden.

Steps Detailed Instructions Comments

Exercise 3 – Create a Triggered Dynamic MRM acquisition method

Task 2. Add/Modify compounds in an existing database

54 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

You select the most abundant transition as the Trigger. The threshold is set
automatically when any acquisition method is updated. You can set the
threshold manually, also.

Table 5 Information for TMRM method for sulfa drugs

Compound Precursor Product Ion Primary Trigger Threshold Collision

Energy

Sulfachloropyridazine 285 155.7 Yes Yes 500 12

Sulfachloropyridazine 285 92.1 Yes No 24

Sulfachloropyridazine 285 108.1 No No 24

Sulfachloropyridazine 285 80.1 No No 60

Sulfachloropyridazine 285 65.1 No No 60

Sulfadimethoxine 311 155.7 Yes Yes 500 16

Sulfadimethoxine 311 107.1 Yes No 28

Sulfadimethoxine 311 245.1 No No 16

Sulfadimethoxine 311 218.1 No No 16

Sulfadimethoxine 311 172.1 No No 32

Sulfadimethoxine 311 140.3 No No 40

Sulfamethazine 279 185.7 Yes Yes 500 12

Sulfamethazine 279 92.1 Yes No 32

Sulfamethazine 279 155.6 No No 16

Sulfamethazine 279 107.7 No No 28

Sulfamethizole 271 155.8 Yes Yes 500 10

Sulfamethizole 271 91.3 Yes No 40

Sulfamethizole 271 177.8 No No 10

Sulfamethizole 271 115 No No 20

Sulfamethizole 271 80.1 No No 40

Sulfamethizole 271 64.8 No No 40

Exercise 3 – Create a Triggered Dynamic MRM acquisition method

Task 3. Create a Triggered Dynamic MRM method from an existing database

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 55

Task 3. Create a Triggered Dynamic MRM method from an existing
database

You can create a Triggered Dynamic MRM method from a database such as the

Pesticide Triggered MRM Database and Library, the Forensics and
Toxicology Triggered MRM Database and Library, or the Veterinary Drug
Triggered MRM Database and Library. These databases can be purchased

from Agilent. You can also copy the information from an Excel spreadsheet,
but that method is not described in this guide.

Steps Detailed Instructions Comments

1 In the Data Acquisition program,

you now import the updated
compounds from the database.
These compounds have optimized
collision energies and also Primary
and Trigger transitions marked.

a Switch to the Data Acquisition

program.

b Open the iiiSulfamix_dMRM2.m

method.

c In the QQQ tab, click the Acquisition

tab. The Scan segments table
contains four rows which are deleted
later.

d Right-click the Scan Segments table

and click Import from Database
Browser. The Database Browser
program opens.

e Mark the Show All Records check

box.

f Mark all of the transitions for the four

sulfa drug compounds. Clear the
check boxes next to any unwanted
compounds.

g Click the Add to Import List button.
h Click the Import List tab.
i Review the Import List table.
j Click the Import button.
k Delete the original compounds from

the Scan segments table.

l Mark the Triggered check box under

Triggered MRM.

• Before you import compounds from
Database Browser, the Scan
segments table contains at least
one row. After importing
compounds from the Database
Browser, you need to remove any
original rows.

• The Scan segments table always
has to have at least one row.

• The triggering information is loaded
from the Database Browser
program even if the Triggered check
box is clear.

• See the online Help for the Data
Acquisition program and the QQQ
Concepts Guide for an explanation
of the other triggering conditions:
Trigger Entrance, Trigger Delay,
and Trigger Window.

Exercise 3 – Create a Triggered Dynamic MRM acquisition method

Task 3. Create a Triggered Dynamic MRM method from an existing database

56 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

2 Save the method to a new method

name, iiiSulfas_TriggerOpt.m,
where iii are your initials.

a Click the Method > Save Method

command.

b Type iiiSulfas_TriggerOpt.m.
c Click the Save button.

Steps Detailed Instructions Comments

Exercise 3 – Create a Triggered Dynamic MRM acquisition method

Task 3. Create a Triggered Dynamic MRM method from an existing database

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 57

3 Review the method in the Dynamic

MRM Viewer dialog box.

a Right-click the Scan segments table

and click Edit DMRM Method. The
Dynamic MRM Viewer dialog box is
opened.

b Type

200 for the Cycle time. This

value is shown in the Acquisition tab.

c Click between the Primaries only

button and the All transitions button
if the Dynamic MRM Statistics
information is not updating. Then,
click the All transitions button.

d Click Close.

• The compounds in Optimizer were

overwritten by the compounds that
you updated in the Database
Browser program.

• You can modify the Cycle time and
see how the Minimum Dwell Time
is changed. If the Minimum Dwell
Time is less than 5 ms, and
especially if it is less than 2 ms,
then signal-to-noise is poor.

• A Dwell Time of 8 ms per transition
is fine.

Steps Detailed Instructions Comments

Exercise 3 – Create a Triggered Dynamic MRM acquisition method

Task 3. Create a Triggered Dynamic MRM method from an existing database

58 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

4 Review the Trigger Thresholds to

verify that they are appropriate.

a Do an injection to make sure that the

Trigger Thresholds are set properly.

b Right-click the Scan segments table

and click Update DMRM Method.

c In the MRM Update Options dialog

box, select True for Update threshold.

d Enter the value for the Percent of

Height for the Trigger Threshold.

e Select the data file that you just

acquired.

f Click OK.

Steps Detailed Instructions Comments

Exercise 4 – Optimize Acquisition parameters

Task 1. Use Optimizer to optimize acquisition parameters

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 59

Exercise 4 – Optimize Acquisition parameters

For this exercise you optimize a mixture of four sulfonamide compounds.

Task 1. Use Optimizer to optimize acquisition parameters

Optimizer helps you optimize acquisition parameters. 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.

To do this task, you first need to create the method iiiSulfamix MRM_10.m in “Task

5. Find optimum collision energy for MRM acquisition”

on page 30. You do not need to

acquire the data file.

Steps Detailed Instructions Comments

1 Start the MassHunter Optimizer

program.

• Double-click the Optimizer icon. . • If you are optimizing peptides, use
the Optimizer for Peptides program.

Exercise 4 – Optimize Acquisition parameters

Task 1. Use Optimizer to optimize acquisition parameters

60 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

Steps Detailed Instructions Comments

Exercise 4 – Optimize Acquisition parameters

Task 1. Use Optimizer to optimize acquisition parameters

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 61

2 Set the optimization parameters. a Click the Optimizer Setup tab.

b Set the Sample introduction method

to Injection (with or without
column).

c Set the range for ramping the Collision

Energy from 0 to 40 V.

d Select a Path for data files to store the

optimization run data.

e Right-click the table on the right and

select Add Method from the shortcut
menu.

f Click the button on the right side of

the Acq Method cell to open the Open
Method dialog box.

g Select the method created in the

previous exercise iiiSulfamix
MRM_10.m and click OK. The Polarity
and Ion Source will be filled in from
the values set in the selected method.

h Check to make sure that the Ion

Source from the method matches the
physical configuration of your
instrument.

i Repeat step e to step h to select

additional methods.

• Fine optimization refines the coarse
ramping values and provides better
optimization but takes longer to
run.

• The data can be displayed later
with MassHunter Qualitative
Analysis program.

Steps Detailed Instructions Comments

Exercise 4 – Optimize Acquisition parameters

Task 1. Use Optimizer to optimize acquisition parameters

62 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

3 Select the precursor ions a Click the Precursor Ion Selection tab.

b Select the Positive ions +H adduct.
c Select the Charge state of 1.
d Set the search priority of the precursor

ions.

e (optional) 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.

• Mark the Use most abundant
precursor ion check box to use the

most abundant precursor ion.

• Clear the Use most abundant
precursor ion check box and use

the Up and Down arrow buttons to
set the search order (ions at the top
of the list are given more priority).

• You can also enter Neutral Losses
to exclude (for example H

2

O).

••

Steps Detailed Instructions Comments

Exercise 4 – Optimize Acquisition parameters

Task 1. Use Optimizer to optimize acquisition parameters

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 63

4 Select the product ions a Click the Product Ion Selection tab.

b Enter a Low mass cut-off value.

Select Mass (m/z) of 60 m/z.

c To exclude certain masses from

consideration, click 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.

d If desired, you can also enter Neutral

Losses to exclude, for example H

2

0.
Enter a formula in the box and click
the button to add it to the list.

• You want to set the Low mass
cut-off value because you do not

want to optimize low mass
non-specific ions. For the sulfa
drugs, significant ions are above 60
m/z.

Steps Detailed Instructions Comments

Exercise 4 – Optimize Acquisition parameters

Task 1. Use Optimizer to optimize acquisition parameters

64 Agilent 6400 Series Triple Quad LC/MS Familiarization Guide

5 Set up a compound list. The

formula for the four Sulfa Drugs
are:

• Sulfamethizole
C

9H10O2N4S2

• Sulfamethazine
C

12H14O2N4

S

• Sulfachloropyridazine
C

10H9O2N4

SCl

• Sulfadimethoxine
C

12H14O4N4

S

a Click the Compound Setup tab.
b Clear the Show results summary

check box above the table while you
set up the compound list.

c Right-click the table and select Add

Compound from the shortcut menu to

add a row to the end of the table.

d Enter

Sulfamethizole as the

Compound Name.

e Enter

Sulfa drugs as the group

name in the Groups column.

f Enter

C9H10O2N4S2 as the

Formula of the compound. The mass
is calculated.

g Enter the Sample Position for the new

compound.

h (optional) Enter an Optimization dwell

time value to set longer or shorter

cycle times.

i Repeat the steps above to add the

other three sulfa drugs to the table.

j Mark the Select columns for the

compounds (rows) to use for
optimization.

k Save the compound list to the

database or to the current project.

• Compounds are global to all
projects. Compound information
such as name, group, formula, and
mass in one project will be
reflected in the entire database.

• 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.

• You can also enter the
monoisotopic mass in the Mass
column instead of the Formula.

Steps Detailed Instructions Comments

Exercise 4 – Optimize Acquisition parameters

Task 1. Use Optimizer to optimize acquisition parameters

Agilent 6400 Series Triple Quad LC/MS Familiarization Guide 65

6 Start the optimization process. • Click the Start Optimization button

( ) on the toolbar

or

• Click the Ion Breakdown Profile
button ( ) on the toolbar.

7 Review results. a Click the Compound Setup tab.

b Mark the Show results summary

check box above the table.

c Review the following values for each

transition ion in the Compound Table:

• Fragmentor

• Collision Energy

d Review the printed optimization

report.

• (optional) Use the MassHunter
Workstation Qualitative Analysis
program to look at the data.

• See the online Help for Optimizer or
the Optimizer Quick Start Guide to
learn how to import optimization
results to acquisition for MRM time
segments.

Steps Detailed Instructions Comments

Agilent Technologies, Inc. 2016

Printed in USA
Revision A, August 2016

*K3335-90207*

K3335-90207

www.agilent.com

In This Book

The exercises in this guide
help you learn to use the
Agilent 6460 Triple Quad
Mass Spectrometer (Model
K6460) system. In this guide,
you acquire data and then
analyze the results using the
MassHunter Qualitative
Analysis program to learn
how to develop an
acquisition method.

For In Vitro Diagnostic Use

The K6460 mass spectrometer is
intended to be used to identify
inorganic or organic compounds in
human specimens by ionizing the
compounds and separating the
resulting ions by means of electrical
field according to their mass.