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
1Check 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.
2Copy 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 1on page 4)
•A 1 ng/μL concentration of the sulfa mix sample (prepared in this step)
1Prepare 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.
2Prepare the sample.
aAdd 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.
bCap the vial and place in a sample location in the autosampler.
3Set 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
4Set 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
1Enter LC parameters appropriate
for sulfa drug mix.
See Table 2.
aDouble-click the Data Acquisition
icon.
bMake 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.
cEnter the LC parameters listed in the
Table 2.
•The Data Acquisition window
appears. See Figure 1on 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
2Enter MS parameters appropriate
for sulfa drug mix and save the
method as iiiMS2Scantest.m,
where iii are your initials.
See Table 3on page 9.
aClick the QQQ tab in the Method
Editor window.
bSelect MS2 Scan from the Scan Type
list in the Time Segments table.
cEnter the other MS parameters as
listed in Table 3. These parameters are
in either the Acquisition or the Source
tabs.
dSave 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
3Acquire 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.
aIf necessary, click View > Worklist to
display the Worklist window.
bClick Worklist > Worklist Run
Parameters. Verify that the
parameters are set properly. Click OK.
cClick Worklist > Add Multiple
Samples.
dType iiisulfamix01.d as the
data file name
eSelect iiiMS2Scantest.m as the
method name.
fClick the Sample Position tab.
gSelect the Autosampler, Well-plate or
Vial Tray.
hIn the graphic, select a single position.
Click OK.
iIn the Worklist window, mark the
check box to the left of the sample.
jClick 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.