Agilent Technologies 6460 Manual de usuario

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.

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.

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