Agilent Technologies 6460 Manual

Masshunter workstation - data acquisition

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Page 1: Table Of Contents
Agilent MassHunter Workstation – Data Acquisition for 6400 Series Triple Quadrupole LC/MS Familiarization Guide For In Vitro Diagnostic Use Before you begin Prepare your system Prepare to acquire data Exercise 1 – Develop an acquisition method Task 1. Enter acquisition parameters and acquire data Task 2.
Page 2 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.
Page 3: Before You Begin
Before you begin Prepare your system 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: •...
Page 4: Prepare To Acquire Data
Triple Quadrupole Column Description Particle Pore Size Part Size Number 6460 RRHD Eclipse Plus 1.8 µm 95Å 959757-902 C18.2.1 mm x 50 mm 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.
Page 5 Before you begin Prepare to acquire data  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)
Page 6: Exercise 1 - Develop An Acquisition Method
Exercise 1 – Develop an acquisition method Task 1. Enter acquisition parameters and acquire data 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.
Page 7 Exercise 1 – Develop an acquisition method Task 1. Enter acquisition parameters and acquire data Table 2 LC parameters for sulfa drug mix (continued) Parameter LC Parameter • Stop Time 2.5 min • Post Time 3.0 min INJECTOR • Inj. Vol. 2.0 µL •...
Page 8 Exercise 1 – Develop an acquisition method Task 1. Enter acquisition parameters and acquire data Figure 1 MassHunter Workstation – Data Acquisition window Steps Detailed Instructions Comments 2 Enter MS parameters appropriate a Click the QQQ tab in the Method for sulfa drug mix and save the Editor window.
Page 9 Exercise 1 – Develop an acquisition method Task 1. Enter acquisition parameters and acquire data 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 •...
Page 10 Exercise 1 – Develop an acquisition method Task 1. Enter acquisition parameters and acquire data Steps Detailed Instructions Comments 3 Acquire data (optional). a If necessary, click View > Worklist to • The Worklist window is tabbed with • Set up a one-line worklist with display the Worklist window.
Page 11: Task 2. Determine Precursor Ion Masses
Exercise 1 – Develop an acquisition method Task 2. Determine precursor ion masses 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.
Page 12 Exercise 1 – Develop an acquisition method Task 2. Determine precursor ion masses Steps Detailed Instructions Comments b Do one of the following: • The figure below shows the default • Select the example data file layout. sulfamix01.d, and click Open. •...
Page 13 Exercise 1 – Develop an acquisition method Task 2. Determine precursor ion masses Steps Detailed Instructions Comments 2 Determine precursor ion masses a In the Chromatogram Results window, • The system displays an averaged for all four peaks. make sure that the Range Select icon spectrum across the peak in the •...
Page 14: Task 3. Find Optimum Fragmentor Voltage For Maximum Response
Exercise 1 – Develop an acquisition method Task 3. Find optimum fragmentor voltage for maximum response 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.
Page 15 Exercise 1 – Develop an acquisition method Task 3. Find optimum fragmentor voltage for maximum response Steps Detailed Instructions Comments b In the Acquisition tab, enter the • With the MS2SIM Scan Type set, a Compound Name and Mass different set of columns appears in (precursor ion mass) for the Acquisition window.
Page 16 Exercise 1 – Develop an acquisition method Task 3. Find optimum fragmentor voltage for maximum response Steps Detailed Instructions Comments 2 Set up and run the worklist a Click the Worklist icon if necessary to • This step is optional because you (optional).
Page 17 Exercise 1 – Develop an acquisition method Task 3. Find optimum fragmentor voltage for maximum response Steps Detailed Instructions Comments 3 Set up a qualitative method to a Click File > Open Data File. • The Qualitative Analysis program view the EIC data automatically. The system displays the Open Data should be open.
Page 18 Exercise 1 – Develop an acquisition method Task 3. Find optimum fragmentor voltage for maximum response Steps Detailed Instructions Comments d If necessary, click Define • The default Method Editor list Chromatograms in the Chromatogram selection after installation is section of the Method Explorer. Integrate (MS).
Page 19 Exercise 1 – Develop an acquisition method Task 3. Find optimum fragmentor voltage for maximum response Steps Detailed Instructions Comments 4 Extract the chromatogram for the a Click the Run button on the Method • You can also click the data file and view the results.
Page 20 Exercise 1 – Develop an acquisition method Task 3. Find optimum fragmentor voltage for maximum response Steps Detailed Instructions Comments 5 Extract the remaining ion a Select File Open Actions from the • The Qualitative Analysis Method chromatograms automatically. General section in the Method Editor lets you define actions to be •...
Page 21 Exercise 1 – Develop an acquisition method Task 3. Find optimum fragmentor voltage for maximum response Steps Detailed Instructions Comments Mark this check box. 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 •...
Page 22 Exercise 1 – Develop an acquisition method Task 3. Find optimum fragmentor voltage for maximum response Steps Detailed Instructions Comments Agilent 6400 Series Triple Quad LC/MS Familiarization Guide...
Page 23 Exercise 1 – Develop an acquisition method Task 3. Find optimum fragmentor voltage for maximum response Steps Detailed Instructions Comments 6 Select the fragmentor voltage that a In the Data Navigator window, • You press the Ctrl key to be able to produces the maximum response highlight the EICs for 271.0 m/z.
Page 24: Task 4. Determine Product Ion Masses
Exercise 1 – Develop an acquisition method Task 4. Determine product ion masses 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.
Page 25 Exercise 1 – Develop an acquisition method Task 4. Determine product ion masses Steps Detailed Instructions Comments 3 Set up a qualitative method to a Click the Open Data File icon in the • The Qualitative Analysis program integrate and extract product ion toolbar.
Page 26 Exercise 1 – Develop an acquisition method Task 4. Determine product ion masses Steps Detailed Instructions Comments n From the Method Explorer in the • These data files contain MS/MS Chromatogram section, click data, so you need to modify the Integrate (MS/MS).
Page 27 Exercise 1 – Develop an acquisition method Task 4. Determine product ion masses Steps Detailed Instructions Comments 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.
Page 28 Exercise 1 – Develop an acquisition method Task 4. Determine product ion masses Steps Detailed Instructions Comments Figure 6 Results for integration and extraction of peak spectra. 5 Run the ‘File Open’ actions on the a Click File > Open Data File. •...
Page 29 Exercise 1 – Develop an acquisition method Task 4. Determine product ion masses Steps Detailed Instructions Comments 6 Identify product ions. a In the Data Navigator, select the TICs • The m/z 155.7 product ion is the • View each set of TICs and and spectra for the 271 m/z precursor most abundant of any product ion spectra individually (e.g., 271...
Page 30: Task 5. Find Optimum Collision Energy For Mrm Acquisition
Exercise 1 – Develop an acquisition method Task 5. Find optimum collision energy for MRM acquisition 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.
Page 31 Exercise 1 – Develop an acquisition method Task 5. Find optimum collision energy for MRM acquisition Steps Detailed Instructions Comments 2 Set up and run the worklist a Click the Worklist tab to make the • This step is optional because you (optional).
Page 32 Exercise 1 – Develop an acquisition method Task 5. Find optimum collision energy for MRM acquisition Steps Detailed Instructions Comments h Click the Overlaid Mode icon, • Compare the colors shown in i Compare peak intensities for each Chromatogram Results with the compound transition in each data file color next to the MRM transition in the Chromatogram Results window.
Page 33: Exercise 2 - Develop A Dynamic Mrm Method From An Mrm Acquisition Data File Or An Mrm Method
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 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...
Page 34 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 Steps Detailed Instructions Comments 2 Create a method for that batch a Click Method >...
Page 35 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 Steps Detailed Instructions Comments • 4 Verify method and then save the a Click Method >...
Page 36: Task 2. Print A Report In The Quantitative Analysis 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 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.
Page 37 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 Steps Detailed Instructions Comments Agilent 6400 Series Triple Quad LC/MS Familiarization Guide...
Page 38: Task 3. Create A Dynamic Mrm Method Using Update Dmrm
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 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.
Page 39 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 Steps Detailed Instructions Comments • The compounds from the data file or quantitation report are automatically added to the Scan segments table. h Verify that each row has a Compound Name.
Page 40: Task 4. Create A Dynamic Mrm Method From An Mrm Method
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 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.
Page 41 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 Steps Detailed Instructions Comments • Agilent 6400 Series Triple Quad LC/MS Familiarization Guide...
Page 42: Exercise 3 - Create A Triggered Dynamic Mrm Acquisition Method
Exercise 3 – Create a Triggered Dynamic MRM acquisition method Task 1. Create a Triggered Dynamic MRM method from a Dynamic MRM method manually Exercise 3 – Create a Triggered Dynamic MRM acquisition method For this exercise you analyze a mixture of four sulfonamide compounds. Task 1.
Page 43 Exercise 3 – Create a Triggered Dynamic MRM acquisition method Task 1. Create a Triggered Dynamic MRM method from a Dynamic MRM method manually Steps Detailed Instructions Comments 2 Change the method to a triggered a Click the Acquisition tab in the QQQ •...
Page 44 Exercise 3 – Create a Triggered Dynamic MRM acquisition method Task 1. Create a Triggered Dynamic MRM method from a Dynamic MRM method manually Steps Detailed Instructions Comments 5 Select the transitions that are the a For each compound, mark the Trigger •...
Page 45: Task 2. Add/Modify Compounds In An Existing Database
Exercise 3 – Create a Triggered Dynamic MRM acquisition method Task 2. Add/Modify compounds in an existing database 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.
Page 46 Exercise 3 – Create a Triggered Dynamic MRM acquisition method Task 2. Add/Modify compounds in an existing database Steps Detailed Instructions Comments 6 Set parameters on the Compound a Click the Compound Setup tab. • For each compound, we are going Setup tab and add additional b Click the Import/Export >...
Page 47 Exercise 3 – Create a Triggered Dynamic MRM acquisition method Task 2. Add/Modify compounds in an existing database Steps Detailed Instructions Comments • To determine product ions in the a Open the SulfamixPI_15.d from “Task • If possible, rearrange the windows Qualitative Analysis program: 4.
Page 48 Exercise 3 – Create a Triggered Dynamic MRM acquisition method Task 2. Add/Modify compounds in an existing database Steps Detailed Instructions Comments 8 Examine the Optimizer Report. a Examine the Collision Energy for each Product Ion. b Print or save the report. As a general rule, as the Product Ions get smaller, the optimal Collision Energy gets larger.
Page 49 Exercise 3 – Create a Triggered Dynamic MRM acquisition method Task 2. Add/Modify compounds in an existing database Steps Detailed Instructions Comments 11 In the Database Browser program, a Mark the Show All Records check • All the transitions that you typed in select the transitions.
Page 50 Exercise 3 – Create a Triggered Dynamic MRM acquisition method Task 2. Add/Modify compounds in an existing database Steps Detailed Instructions Comments 12 In the Database Browser program, a In the Set top ranked transitions as • The program automatically selects automatically select the Primary primary box, enter 2.
Page 51 Exercise 3 – Create a Triggered Dynamic MRM acquisition method Task 2. Add/Modify compounds in an existing database Steps Detailed Instructions Comments 13 Review the Primary transitions and • Review each compound. Change the • The program selected the most Trigger transitions.
Page 52 Exercise 3 – Create a Triggered Dynamic MRM acquisition method Task 2. Add/Modify compounds in an existing database Steps Detailed Instructions Comments 14 Review the Import List table on a Click the Add to Import List button. • In this example, you are importing the Import List tab.
Page 53 Exercise 3 – Create a Triggered Dynamic MRM acquisition method Task 2. Add/Modify compounds in an existing database Steps Detailed Instructions Comments 16 Save the new compound • Click the File > Save Compounds • You cannot see these results by parameters to the database.
Page 54 Exercise 3 – Create a Triggered Dynamic MRM acquisition method Task 2. Add/Modify compounds in an existing database Table 5 Information for TMRM method for sulfa drugs Compound Precursor Product Ion Primary Trigger Threshold Collision Energy Sulfachloropyridazine 155.7 Sulfachloropyridazine 92.1 Sulfachloropyridazine 108.1 Sulfachloropyridazine...
Page 55: Task 3. Create A Triggered Dynamic Mrm Method From An Existing Database
Exercise 3 – Create a Triggered Dynamic MRM acquisition method Task 3. Create a Triggered Dynamic MRM method from an existing database 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.
Page 56 Exercise 3 – Create a Triggered Dynamic MRM acquisition method Task 3. Create a Triggered Dynamic MRM method from an existing database Steps Detailed Instructions Comments 2 Save the method to a new method a Click the Method > Save Method name, iiiSulfas_TriggerOpt.m, command.
Page 57 Exercise 3 – Create a Triggered Dynamic MRM acquisition method Task 3. Create a Triggered Dynamic MRM method from an existing database Steps Detailed Instructions Comments 3 Review the method in the Dynamic a Right-click the Scan segments table • The compounds in Optimizer were MRM Viewer dialog box.
Page 58 Exercise 3 – Create a Triggered Dynamic MRM acquisition method Task 3. Create a Triggered Dynamic MRM method from an existing database Steps Detailed Instructions Comments 4 Review the Trigger Thresholds to a Do an injection to make sure that the verify that they are appropriate.
Page 59: Exercise 4 - Optimize Acquisition Parameters
Exercise 4 – Optimize Acquisition parameters Task 1. Use Optimizer to optimize acquisition parameters 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...
Page 60 Exercise 4 – Optimize Acquisition parameters Task 1. Use Optimizer to optimize acquisition parameters Steps Detailed Instructions Comments Agilent 6400 Series Triple Quad LC/MS Familiarization Guide...
Page 61 Exercise 4 – Optimize Acquisition parameters Task 1. Use Optimizer to optimize acquisition parameters Steps Detailed Instructions Comments 2 Set the optimization parameters. a Click the Optimizer Setup tab. • Fine optimization refines the coarse b Set the Sample introduction method ramping values and provides better to Injection (with or without optimization but takes longer to...
Page 62 Exercise 4 – Optimize Acquisition parameters Task 1. Use Optimizer to optimize acquisition parameters Steps Detailed Instructions Comments 3 Select the precursor ions a Click the Precursor Ion Selection tab. • Mark the Use most abundant b Select the Positive ions +H adduct. precursor ion check box to use the c Select the Charge state of 1.
Page 63 Exercise 4 – Optimize Acquisition parameters Task 1. Use Optimizer to optimize acquisition parameters Steps Detailed Instructions Comments 4 Select the product ions a Click the Product Ion Selection tab. • You want to set the Low mass b Enter a Low mass cut-off value. cut-off value because you do not Select Mass (m/z) of 60 m/z.
Page 64 Exercise 4 – Optimize Acquisition parameters Task 1. Use Optimizer to optimize acquisition parameters Steps Detailed Instructions Comments 5 Set up a compound list. The a Click the Compound Setup tab. • Compounds are global to all formula for the four Sulfa Drugs b Clear the Show results summary projects.
Page 65 Exercise 4 – Optimize Acquisition parameters Task 1. Use Optimizer to optimize acquisition parameters Steps Detailed Instructions Comments 6 Start the optimization process. • Click the Start Optimization button ) on the toolbar • Click the Ion Breakdown Profile button ( ) on the toolbar.
Page 66 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.

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