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LCquan 2.5.6 Quantitative Analysis User Guide

1. LCquan User Guide Thermo Scientific Explore View Figure 6 A LCquan Steroids Workbook File Zoom View Apps GaH Options Help Leja O 1 Introduction Introduction to Quantitative Analysis with LCquan The Explore view allows you to display a multi peak chromatogram for a single sample and then to experiment with peak detection and integration parameters to see how they affect the chromatogram In the Explore view you determine the optimum parameters to be used in the Processing Method LCquan can also generate a peak list from which you can generate quan components to be used in the Processing Method The Create Explore Method page of the Explore view is shown in Figure 6 000000 00 Explore view Create Explore Method page showing a multipeak chromatogram DEX Identification a Chromatogram Definition Genesis Peak Integration Limit peaks Acquisition Sequence History Detector Smoothing Peak Detection Algorithm Select top peaks MS S Genesis v Advanced Enable Trace TIC z S N Threshold 0 5 TREA C Valley Detection Enabled dth teea 0 0 Ne Fit C Constrain Peak Width Rel peak height threshold be Peak Ht Enable Range min Reg sora 100 0 0 999 9 Save
2. A LCquan Steroids Workbook Eek File Zoom View Apps Options Help HGBHEGEELS YR COCOGCTCOO Hi BO Instruments Review All Results review standards QCs blanks and unknowns Acqisien J Fiename integration Type ISTO Response Specified Cone Caeuted Gone Peak stus wom orsa DEES leoxycorticosterone Explore 3 i methyltestosterone Quantitare 2 mes Method Settings 905012 704901 1284 progesterone j4 sampleD Method Settings 278722 691760 0 403 NA 0 723 NA 15 sample Method Settings 489300 716881 0 683 NA 1 224 NA x 4 gt AI A Standards Cs Blanks Unknowns e il E oe Selected Chromatogram v 1 gt 8 isto he Chromatogram yj dea Calibration Curve v campleA miz 267 10 267 10 309 10 309 10 SM 7 RT 0 13 113 NL 4 32E5 sampled miz 267 10 267 10 285 20 265 20 SM 7 RT 1 52 2 52 NL 1 02E5 hydrocortisone F Full ms2 363 30 150 00 375 00 F Full ms2 303 30 100 00 310 00 Y 0 557511 R 2 09995 W Equal 100 RT 0 69 100 AT 2 02 7 30 6 80 70 5 z is z 4 g 3 2 50 D i i a 40 30 2 20 1 10 173 238 247 0 s o 2 4 6 a 0 42 02 03 04 05 06 OF os 09 10 WwW 16 18 20 22 24 Time min Time min ng For Help press F1 10 25 2005 9 23 A Thermo Scientific LCquan User Guide 115 6 Processing the Raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Figure 55 Peak Information dialog box
3. Sample Type File Name Level 1 Standard steroids02 cal04 2 Standard steroids03 cal03 3 Standard steroids04 cal02 4 Standard steroids05 cal01 5 Unknown samplea 6 Unknown sampleb 7 Unknown samplec 8 Unknown sampled 9 Unknown samplee 10 Standard steroids13 cal04 11 Standard steroids14 cal03 12 Standard steroids15 cal02 13 Standard steroids16 cal05 The Acquisition Sequence should now look like Figure 16 34 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View Figure 16 Edited Acquisition Sequence for the steroids example 4 LC quan Steroids Workbook Dek _Ele Change Actions Yiew Apps Options Help GSH E44 GL gt 000000000 iii Acquisition Sequence History FileName steroids02 Study Example Study 76 Sample Name PrevRow Row 1 NextRow Clear All __ Sample Type FileName Levei carte Sample Path Inst M 2 Standard steroids03 cal03 C XcaliburiQuanRootlExample Study C Xcalibur QuanRoo a Standard steroids04 cal02 C XcaliburiQuanRoot Exemple Study C Xcalibur QuanRoo 4 Standard steroids05 cal01 C XcaliburiQuanRootlExample Study C Xcalibur QuanRoo 5 Unknown samplea NA sample006 C Xcalibur QuanRootExample Study C XcaliburiQuanRo
4. Click on Method button Click on Method button y Create Method Page Create Explore Method Page Click on Sequence button Click on Review button Create Sequence Page Survey Page Click on Survey button Review All Results Page Click on Review All button Review the Explore Results Page Review Reports Page Click on Reports button Thermo Scientific LCquan User Guide 125 A LCquan Flow Diagrams 126 Figure 59 Acquisition view flow diagram LCquan Acquisition Flow Diagram Base acquisition ona Import Sequence previously saved file Create New Create new method Generate Sample File Dialog Box r in New Acquisition Wizard Acquisition Sequence Names Page in New Acquisition Wizard Click on Click on Next then Next click on Finish Sample Information Page Click on Next Vial Positions Page Click on Next Add Standards Page Click on Next Add QCs Page Click on Next Component Name Page Click on Next then Acquisition View iaa i Setup Sequence lt User Label Page Page Define Levels Click on Acquire button in viewbar Acquisition View Run Sequence Dialog Box Click on OK Acquisition View _ _____ Quantitate Status Page Automatically enter View Processing View if Process After Acquisition was selected in Run Sequence dialog box LCquan User Guide Thermo Scientific A LCquan Flow Diagrams Figure 60 Q
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6. i ly W gt CBOOSOCOOC Ee Example Study 76 Acquisition Sequence History Sequence information for multiple sequence lists sarmpio te _Fiewarme tewet _calfie Senge sid Sith i inst 4 Standard 5 Standard e Unknown Unknown jae Unknown 9 Unknown mo Standard 12 Standard 13 Standard 44 Standard 15 Standard steroids002 cal02 C XcaliburvQuanRootiExample Study C Xcalibur QuanRoo steroids003 cal03 i 7 E 5 C XcaliburQuanRootiExample Study C Xcalibur QuanRtoo steroids004 call4 C XcaliburvQuanRootiExample Study C Xcalibur QuanRoo steroids005 cal05 C XcaliburQuanRootiExample Study C XcaliburiQuanRoo steroids006 NA sample006 C Xcalibur QuanRoot Example Study C XcaliburiQuanRoo steroids007 NA sample007 C Xcalibur QuanRootExample Study C Xcalibur QuanRoo steroids008 NA sample008 C Xcalibur QuanRoot Example Study C Xcalibur QuanRoo steroids009 NA sample009 C XcaliburiQuanRootiExample Study C Xcalibur QuanRoo Unknown steroids010 NA sample010 C XcaliburiQuanRoot Example Study C XcaliburiQuanRoo steroids011 cal01 C XcaliburiQuanRootiExample Study C XcaliburiQuanRoo steroids012 cal02 C XcaliburtQuanRoot Example Study C Xcalibur QuanRoo steroids013 cal03 C XcaliburvQuanRootiExample Study C XcaliburlQuanRoo steroids014 cal04 C XcaliburiQuanRootlExa
7. A calibration standard is a sample containing known amounts of all target components The purpose of a standard is to measure the response of the instrument to the target components so that a calibration curve can be generated for each component An unknown sample is one containing unknown amounts of the target components LCquan performs quantitative analysis on any sample defined as an unknown sample LCquan User Guide 9 1 Introduction Introduction to Quantitative Analysis with LCquan QCs A QC sample contains a known amount of one or more specific target compounds QC samples are placed in the sequence so that quantitative analysis results can be tested for quality assurance purposes After the QC sample is analyzed the measured quantity is compared with the expected value and an acceptability range The quantitative analysis of a QC sample is classified as passed if the difference between the observed and expected quantities is within the user defined tolerance A QC sample is classified as failed if the difference between the observed and expected quantities is outside the defined tolerance Blanks A blank sample contains no target components but might contain an ISTD when the internal standard quantitative analysis technique is being used The analysis of a blank sample allows you to confirm that there are no residual components in the solvent system that can cause erroneous results Introduction to Quantitative Analysis with LCq
8. LCquan User Guide 5 1 Introduction Quantitative Analysis Techniques 6 LCquan User Guide Figure 1 Integrated chromatographic peak 10 80 60 Relative Intensity 20 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 0 Time min Quantitative Analysis of samples containing unknown amounts of the target component is achieved by first calculating the peak area or height and then computing and applying the appropriate response to the equation derived from the calibration curve This provides an estimate of the amount of the unknown component The precision of the measurement depends on the quality and to a lesser extent the quantity of the calibration data The detection limit of the quantitative analysis method is the lowest concentration of analyte in a sample that can be detected but not necessarily quantitated as an exact value The lower and upper quantification limits are the lowest and highest concentrations of analytes in a sample that can be measured with an acceptable level of accuracy and precision In an analytical method the highest concentration calibration standard defines the upper quantification limit The quantification range is the range of concentration between the lower and upper quantification limits including these limits that can be reliably and reproducibility quantified with acceptable levels of accuracy and precision through the use of a concentration response relationship There are two basic quantitative analys
9. Thermo Scientific After you create or import an Acquisition Sequence you can edit it by using the Acquisition view Setup Sequence page You can change the Acquisition Sequence in the following ways e By editing it directly e By modifying its arrangement e By changing the user labels that appear in the Sequence Header Editing the Acquisition Sequence Directly You can edit the Acquisition Sequence by manually changing the entry in any of the cells in the Sequence table To change the sample type or the calibration or QC level select a new option from the list box in the Sequence table For example to change the sample type for a particular sample click in the cell in the Sample Type column click on the arrow and then select the new sample type from the list To change any other entry simply type in the appropriate text box For example to change the file name for a particular sample click in the cell in the File Name column delete the old name and type the new name In addition instead of typing the injection volume for a sample you can specify that LCquan is to use the injection volume from the Instrument Method for the autosampler To do this select a sample row or rows and then right click anywhere in the Sequence table to display the shortcut menu Choose Get Injection Volume From AS In the rows that you selected the cells in the Injection Volume column now display From AS LCquan User Guide 39 2 Preparing to Perf
10. Thermo Scientific LCquan User Guide 15 1 Introduction Introduction to Quantitative Analysis with LCquan 16 LCquan Folder Structure LCquan User Guide When you start a new quantitative analysis project LCquan creates a hierarchical folder structure for the project on the hard drive See Figure 8 Figure 8 Folder structure created by LCquan for a quantitative analysis project 3 C Example Study 6 Example Workbook C Exports C Imports C Rawfiles C Temp The top most level in the file structure is the study folder This folder is used to organize one or more workbook folders Each workbook folder holds all of the information used by LCquan for an individual quantitative analysis project The workbook folder contains the LCquan file gz file the Instrument Method file s meth file and the audit log files In addition the workbook folder contains the following folders Exports Imports Rawfiles and Temp The Exports folder stores copies of all files that are exported from LCquan such as report files The Imports folder stores a copy of legacy files that you import into the workbook such as Instrument Method files or sequence files The Rawfiles folder contains acquired data files raw files as well as any imported raw data files The Temp folder contains backup and temporary files used by LCquan When you create a new project LCquan requires you to name the study folder and the workbook fol
11. 09 Retie Intensity 8 8 100 30 8 3 g 3 8 8 steroidst6 TIC SM 7 RT 1 60 2 35 NL 3 59E5 F c Full ms2 303 30 40 00 100 00 310 00 Area Ratio FT 1 37 2 25 mT T LAA nnd Ww 18 13 20 24 Time min hydrocortisone Y 0 0293758 1 14164 X R 2 0 9996 Wi Equal 14 12 10 For Help press F1 Thermo Scientific _ NOT SAVED 10 19 2005 3 45 Pr LCquan User Guide 113 6 Processing the Raw Files and Reviewing the Analytical Results Reviewing All Results Reviewing the Unknowns To review and rework the results for the unknowns 1 Click on the Unknowns tab at the bottom of the Result List to display the peak integration results for the unknown samples 2 In the Component List select the internal standard methyltestosterone 3 Inspect each file in the Result List and check for peak detection and integration problems 4 Inspect the chromatogram in the Chromatogram pane for each file a Ensure that LCquan found the peak b Ensure that the shaded area accurately represents the contribution of the component to the chromatogram e Ifyou want to modify the peak detection and integration settings go to the topic Modifying the Peak Detection and Integration Settings on page 109 e Ifyou want to integrate a peak manually go to the topic Integr
12. Sample Type Sample ame integration Typa Response ISTO Response peated Cone 8 hydr e deoxycorticosterone methyltestosterone 2 steroids03 Standard Method Settings 7031020 2314238 3 038 2 500 progesterone a steroids04 Standard Method Settings 3666547 2595302 1413 1 250 4 steroids05 Standard Method Settings 1782495 2403939 0741 0 630 a steroids13 Standard Method Settings 13449804 2361619 5 695 5 000 je steroids14 Standard Method Settings 7160845 2394666 2 990 2 500 steroids15 Standard Method Settings 3574151 2567027 1 392 1 250 ja __ steroidst6 Standard Method Settings 34771261 2530482 13 741 12 000 1ra Chromatogram Y ISTD v Chromatogram Vv Rette heast Time min Calibration Curve Retatue heist oT steroidc02 TIC SM 7 RT 1 63 2 38 NL 3 30E5 F c Full m2 303 31 00 310 00 179 184 18 13 20 Es Time min 22 Area Ratio Y 0 0296878 1 12704 R 2 0 9926 WW Equal 14 12 10 hydrocortisone For Help press F1 Thermo Scientific Moved Integration Marker NOT SAVED 10 19 2005 2 46 PI LCquan User Guide 111 6 Processing the Raw Files and Reviewing the Analytical Results Reviewing All Results Reviewing All Results You use the Review All Re
13. The Acquisition Sequence History lists every Acquisition Sequence that was submitted to the acquisition queue in the life of the workbook It provides information on each sequence including the operator the start and end times comments and the status of the sequence as well as the calibration and QC level data While LCquan is acquiring data the history is dynamically updated with samples that have been fully acquired To display the history click on the Acquisition History tab The Chromatogram pane of the Acquisition Status view allows you to monitor chromatograms for each of the defined components plus up to ten user defined chromatograms in real time up to five are visible at one time It can also display data from an existing raw file The Spectrum pane allows you to monitor the mass spectrum in real time or to view the mass spectrum of a selected raw file Using the Chromatogram Pane 48 LCquan User Guide You specify the properties of the chromatograms that LCquan displays by using the Realtime Display Settings dialog box To open the Realtime Display Settings dialog box choose Options gt Realtime Display Settings See Figure 22 This dialog box consists of two parts the Options group box and the User Defined traces group box The settings in the Options group box allow you to specify the types and number of chromatograms to display the time range information for the chromatograms and whether or not to display the spectrum t
14. _locked_nnn to the original workbook name where nnn is the number of the locked workbook For example the first time that you create a locked workbook from the workbook Trial1 LCquan names the locked workbook Triall_locked_001 The second time that you lock the workbook Triall LCquan names the locked workbook Triall_locked_002 You can create up to 999 locked versions of a workbook You cannot unlock a locked workbook When you open a locked workbook LCquan displays Locked in the title bar next to the workbook name and in the status bar Saving the Workbook and Exiting LCquan You can save the workbook at any step in the LCquan procedure This allows you to exit LCquan and reenter at the same place at a later time When you reopen a workbook LCquan displays the page and view that were open when you exited the workbook To save the workbook and exit LCquan 1 Choose File gt Save to save the current workbook If you want to save the workbook with a different study or workbook name choose File gt Save As The Save As Wizard appears to guide you through the procedure 2 Choose File gt Exit to exit LCquan Note If you acquire data LCquan will not let you exit the program unless you save the workbook Thermo Scientific LCquan User Guide 121 LCquan Flow Diagrams This appendix contains a set of flow diagrams for LCquan for you to use as a reference The flow diagrams show step by step progressions through the di
15. Identification Calibration Chromatogram Definition ICIS Peak Integration Peak Identification hydocottisone Detector Smoothing Peak Detection Algorithm MOTOR eOe MS 1 ES Advanced Highest Peak Min Peak Height S N _ Progesterone Tce O Neas Baseline Window 40 TIC v v gt a lon ratio confirmation Mass m 2 Area Nojse Factor 5 C Enabled lon ratio using Area so Fonie 0 mo r 10 v Constrain Peak Width Retention Time Peak Ht 5 0 Expected min 1 00 Window sec 30 0 Tailing Factor 1 0 Use as RT Ref View Width min 0 75 Adjust Using Apply Cancel Save As Default Add dette __ Rename romatogram ectrum he d gt a Chromatog dra Spect steroids04 TIC RT 0 51 1 26 NL 1 06E6 steroids04 79 RT 0 89 NL 3 67E3 T c Full ms2 363 30 40 00 150 00 375 00 _ as TA 0 68 ee 327 21 90 90 80 80 704 70 q e T 60 5 S z 004 50 2 40 2 40 30 30 204 20 104 10 f 142 146 122 P 08 a7 08 09 10 44 12 150 200 250 300 350 Time min For Help press F1 O NOT SAVED 11 2 2005 1 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Specifying the Component Identification and Integration Parameters Specifying the Component Identification and Integration Parameters LCquan needs component identification information to associate the target compounds and internal standard with chromatogram peaks The following topics describe
16. Modifying the Peak Detection and Integration Settings 109 Integrating Peaks Manually ny suse ated tirinareue ae pen lt 110 Reviewing All Restltsnosd sates Gite hie wee eae rene 112 Opening the Review All Results Page 0 0 eee eee eee 112 Reviewing the Unknowns aden ieetie apie ei dda Ride rnnr 114 Creating and Reviewing Reports o c i Sok cacead oes eee SE bee OS 116 Locking the Workbook steric cckseaathit bylivenatedale bowie dclardoacayeediaergcaneyeed ate 121 Saving the Workbook and Exiting LCquan 0 e eee eee 121 Appendix A LCquan Flow Diagrams 002 0c eee e cece eee eee 123 LCquan User Guide v Preface About This Guide LCquan is part of the Xcalibur mass spectrometry data system This user guide describes how to use LCquan to perform quantitative analysis of compounds Related Documentation The following LCquan manuals are available on the LCquan software CD as PDF files e LCquan Administrator Guide describes how to configure the software for compliance with 21 CFR Part 11 e LCquan User Guide describes how to use LCquan to perform quantitative analysis of compounds To view the LCquan manuals Go to Start gt Programs gt Xcalibur gt Manuals gt LCquan The Help contains an LCquan Tutorial that provides an overview of how to use LCquan with an example data set To open the LCquan Help From the LCquan window choose Help gt LCquan Help
17. T samplee steroids02 T steroids03 steroids04 7 steroidsOS steroids13 steroids14 steroids15 File name steroids 7 raw samplea raw sampleb raw W Files of type Raw Files raw a E 7 Ensure that the Import Instrument Method Acquisition Sequence and Processing Parameters check box is cleared and click on Next 8 Click on Finish LCquan creates the new study and workbook folders and imports the raw data files into the Rawfiles subfolder After you create a new workbook LCquan opens the Instrument Setup view Because there are no Instrument Methods for the configured devices yet LCquan creates default methods You use the raw files that you just imported to complete the steroids quantitative analysis described in this manual Therefore you do not need to create an Instrument Method or acquire your own data If you want to continue directly with the quantitative analysis of steroids procedure skip the next several sections in this chapter and continue with Creating an Acquisition Sequence for the Steroids Example on page 30 24 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Instrument Setup View Reviewing the Instrument Setup View The Instrument Setup view allows you to define a set of experimental parameters for each configured instrument These parameters include the operating settings for the ins
18. To locate a particular topic use the Help Contents Index or Search panes Thermo Scientific LCquan User Guide vii Preface Special Notices Special notices include the following IMPORTANT Highlights information necessary to avoid damage to software loss of data invalid test results or information critical for optimal performance of the system Note Highlights information of general interest Tip Helpful information that can make a task easier Contacting Us There are several ways to contact Thermo Fisher Scientific To contact Technical Support Phone 800 685 9535 Fax 561 688 8736 E mail TechSupport C MS thermofisher com Knowledge base www thermokb com Find software updates and utilities to download at www mssupport thermo com To contact Customer Service for ordering information Phone 800 532 4752 Fax 561 688 8731 Web site www thermo com finnigan To suggest changes to documentation or to Help e Fill out a reader survey online at www thermo com Icms techpubs e Send an e mail message to the Technical Publications Editor at techpubs finnigan lcms thermofisher com viii LCquan User Guide Thermo Scientific Introduction This chapter describes some of the basic principles and terminology of quantitative analysis and provides a brief overview of quantitative analysis with LCquan This chapter contains the following sections Contents e About Quantitative Analysis e Considering the Variables o
19. Zoom View Apps Options Help Reviewing All Results y a GeaHARLLEAGRCCOSCOCOO KE GO _ tnctuments _ Review All Results review standards QCs blanks and unknowns Filename Sample Type Sample Hame Integration Type Response ISTD Response Response Ratio Specified Conc 1 steroids02 Standard Method Settings 13775248 2435216 5 657 5 000 fl retiyitestosterone 2 steroids03 Standard Method Settings 7031020 2314238 3 038 2 500 progesterone 3 steroids04 Standard Method Settings 3666547 2595302 1 413 1 250 4 steroids05 Standard Method Settings 1782495 2403939 0 741 0 630 5 steroids13 Standard Method Settings 13449804 2361619 5 695 5 000 6 samplea Unknown Method Settings 13889320 2382340 5 830 NA 7 sampleb Unknown Method Settings 6882603 2370762 2 903 NA 8 f 9 sampled Unknown Method Settings 1899556 2391928 0 794 NA 10 samplee Unknown Method Settings 3575212 2463101 1 452 NA 11 steroids14 Standard Method Settings 7160845 2394666 2 990 2 500 12 steroids15 Standard Method Settings 3574151 2567027 1 392 1 250 13 Selected v Chromatogram Vv dda is Kale ISTD Chromatogram v 4 gt a Calibration Curve Rette heast steroidst6 TIC SM 7 PT 0 29 1 04 NL 1 18E7 F Full mz2 363 30 40 00 150 00 375 00 PT 0 67 06 o7 Time min 08
20. and click Apply to display the filtered chromatogram Note that progesterone corresponds to the peak named Peak 3 151 in the peak list 5 Select Smoothing Zand Peak Detection Algorithm Genesis and click Apply Notice the effect on the progesterone peak Note The Explore view is where you experiment with the peak detection and peak integration parameters Refer to the Help for descriptions of the peak detection and peak integration parameters 6 Clear the Filter box click Apply Go on to the next topic Reviewing the Explore Results 58 LCquan User Guide Thermo Scientific Reviewing the Explore Results 3 Exploring Your Data Reviewing the Explore Results In this section you review the filtered chromatograms and mass spectra of the steroids The unfiltered chromatogram is shown in Figure 28 To review the Explore results hydrocortisone mass spectrum Select Multi Peak Plot in the right side pane Click on the Review button LCquan displays the Review the Explore Results page See Figure 28 Click on the top chromatogram peak hydrocortisone in the multipeak plot LCquan displays the product ion mass spectrum at the maximum intensity Record the mass to charge ratios of the first several ions of highest intensity in the Figure 28 Explore view Review the Explore Results page showing the unfiltered chromatogram for the steroids Workbook AY LC quan Steroids File Zoom Yiew Apps Options
21. causes interference See the online Help for further information about these advanced parameters 7 Click on Apply to apply the changes to the sample that is currently selected in the Result List or click on Apply To All to apply the changes to all of the samples on the current page of the Result List for example to all samples on the Standards page 8 When you are finished click on OK to close the dialog box If you changed any parameters since you last clicked on the Apply button or the Apply To All button clicking on OK will apply the new changes to the sample selected in the Result List When you modify the peak detection and integration parameters LCquan displays User Integration in the Integration Type column of the Result List To revert back to the original peak detection and integration parameters from the Processing Method do the following In the Integration Type column of the Result List click in the cell containing the words User Integration for the sample that you want to change Then click on the down arrow and select Method Settings from the drop down list Thermo Scientific LCquan User Guide 109 6 Processing the Raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Integrating Peaks Manually You can manually change the baseline and the starting and ending points of a peak in either of two ways e By clicking and dragging the starting and ending points To move a start
22. click on the Edit button to return to the Create Sequence Review and Edit page Figure 46 Raw files for the steroids example and their calibration levels 2 Standard steroids04 cal02 3 Standard steroids15 cal02 steroids03 cal03 steroids14 cal03 stercids02 cal04 Standard steroids13 cal04 steroids16 cal05 a Unknown samplea NA Unknown sampleb NA Unknown samplec NA Unknown sampled NA Unknown samplee NA When you are satisfied that the Processing Sequence for the steroids example is correct do one of the following e Ifyou want more information on Processing Sequences before continuing go to the next sections Further Information Additional Ways to Define the Processing Sequence on page 95 and Further Information Editing the Processing Sequence on page 97 e Ifyou want to process the steroids raw data files go to Chapter 6 Processing the Raw Files and Reviewing the Analytical Results 94 LCquan User Guide Thermo Scientific 5 Creating a Processing Sequence Further Information Additional Ways to Define the Processing Sequence Further Information Additional Ways to Define the Processing Sequence Usually instead of creating a new Processing Sequence it is more convenient to use an existing Processing or Acquisition Sequence as your Processing Sequence You can do this in either of two ways as follows e Importing an Existing Sequence e Copying the Acquisition Sequence Importing an Existi
23. select the Generate Selected Reports check box If you do not want LCquan to process the data after it is acquired clear the Process After Acquisition check box 6 Specify any optional Startup or Shutdown Instrument Methods If you want the Startup Method to be run before the sequence is run select the Use Startup Method check box If you want the Shutdown Method to be run after the sequence is run select the Use Shutdown Method check box If you do not want these methods run clear the check boxes Thermo Scientific 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View 7 Specify any optional pre acquisition or post acquisition programs a In the Pre Acquisition text box specify the pre acquisition program You can use the Browse button to locate the program b In the Post Acquisition text box specify the post acquisition program You can use the Browse button to locate the program c Specify whether to run these programs synchronously in series or asynchronously in parallel with the data acquisition e Ifyou want to run these programs synchronously select the Run Synchronously Pre Acquisition check box and the Run Synchronously Post Acquisition check box e Ifyou want to run these programs asynchronously clear the Run Synchronously Pre Acquisition check box and the Run Synchronously Post Acquisition check box 8 Specify the status of the system after data acqui
24. solution stability injection reproducibility and matrix interference lead to lower precision levels in the external standard method than in the internal standard method Figure 2 Calibration curve generated by using an external standard 900000 Response for Unknown Sample 400000 4 ge 300000 200000 Response for Target Compound 100000 4 Amount A in Unknown Sample 0 T T T T 1 0 20 40 60 80 100 Amount of Target Compound A LCquan User Guide 7 1 Introduction Quantitative Analysis Techniques Using Internal Standards for Quantitative Analysis LCquan User Guide An internal standard ISTD is a component that is added to a sample to act as a response reference for one or more non ISTD components in the sample The concentration or amount of an ISTD in any standard or unknown sample remains constant Because quantitative mass spectrometric analysis usually involves multiple steps the total error in the analysis results from the accumulation of the errors at each step In general sample handling errors account for a larger fraction of the total error than do mass spectrometer errors Fortunately the internal standard method can reduce both sources of error For example internal standards can correct for variations in a component s peak area that are caused by the following e Injection irreproducibility e Changes in analyte solution volume e Matrix and coeluter interference both suppression
25. 30 40 100 00 310 00 ina Peak Ht 5 0 Enable Range min Tailing Factor 10 of highest peak 100 0 0 999 9 Apply Cancel Save As Defaut gt a Chromatogram Mld gt a Peak List steroids04 TIC SM 7 RT 0 01 3 79 NL 3 73E5 eteroidso4 F c Full ms2 303 30 40 00 100 00 310 00 ml Peak Name Apex RT Area of Largest Area of Total Area 100 RT 1 99 1 Peak 1991 1 991 2605126 100 000 100 000 2 s0 e e 804 B E 5 704 J B gt EJ 604 a J 10 E ia 3 s04 A 2 4 12 k 13 40 LES e E 14 304 H 23 hz J 18 J 19 105 o J 49 z o 22 05 1 0 15 20 25 3 0 35 z Time min 24 w For Help press F1 Thermo Scientific NOT SAVED 11 17 2005 5 07 PI LCquan User Guide 57 3 Exploring Your Data Identifying Components 2 Select the scan filter for deoxycorticosterone parent ion mass m z 331 Filter c Full ms2 331 30 40 100 00 350 00 and click Apply to display the filtered chromatogram Note that deoxycorticosterone corresponds to the peak named Peak 1 392 in the peak list 3 Select the scan filter for hydrocortisone parent ion mass m z 363 Filter c Full ms2 363 30 40 150 00 375 00 and click Apply to display the filtered chromatogram Note that hydrocortisone corresponds to the peak named Peak 0 675 in the peak list 4 Select the scan filter for progesterone parent ion mass m z 315 Filter c Full ms2 315 30 40 100 00 320 00
26. 4 Click on Finish to exit the New Processing Method Wizard The Create Sequence Select Files page of the Quantitate view opens You are now ready to begin defining the Processing Sequence Figure 42 New Processing Sequence Wizard Welcome page New Processing Sequence Wizard Welcome to the New processing sequence wizard LCqua m 7d 5 This wizard helps you setup the processing sequence A New Era in Mass Spectrometry ELECTRON CORPORATION 88 LCquan User Guide Thermo Scientific 5 Creating a Processing Sequence Associating Raw Files with Sample Types Associating Raw Files with Sample Types When you use LCquan for quantitative analysis you normally do not need to create a new Processing Sequence in the manner described below Instead you copy the Acquisition Sequence from the current workbook to use as the Processing Sequence or you import an existing sequence from another workbook or legacy file These techniques allow you to define your Processing Sequence quickly and conveniently For more on these techniques refer to Further Information Additional Ways to Define the Processing Sequence on page 95 The steroids quantitative analysis example outlined in this manual is a special case You do not need to create an Acquisition Sequence because the raw data already exists For practice you created an Acquisition Sequence in the topic Creating an Acquisition Sequence for the Steroids Example on page 30 Th
27. As Default Chromatogram q gt a Spectrum he sampleD TIC SM 7 RT 0 01 3 79 NL 4 81E5 sampleD 162 RT 1 99 NL 5 99E4 T Full ms2 303 30 40 00 100 00 310 00 1004 RT 0 68 100 267 24 s4 90 4 E 80 kE 70 J 2 604 T 60 E J g s3 3 50 d S 2 E iE e 40 304 30 s 20 104 10 J od 0 25 0 20 Time min 100 150 200 miz 250 300 For Help press F1 Thermo Scientific NOT SAVED 10 25 2005 3 39 PM LCquan User Guide 13 1 Introduction Introduction to Quantitative Analysis with LCquan 14 Quantitate View In the Quantitate view see Figure 7 LCquan steps you through a procedure for batch processing raw data files during quantitative analysis The LCquan window incorporates an integrated calibration curve peak integration and results view that allows you to review and rework standards QCs and unknowns interactively in one window You can also enter the Quantitate view while you are acquiring data to process the data acquired to date Figure 7 SN L quan Steroids Workbook Eile Zoom View Apps Options Help Quantitate view Review All Results page ek 1 ToL G HEG LES PR CCOSCSCSOO Ht OO _ tnstuments _ Review All Results review standards QCs blanks and unknowns Filename Sample Type Sample Hame Integration Ty
28. EEk Fie Zoom Yiew Apps Options Help beHa EE Survey review standards and QCs __fitewame Semple ype Sample Nome integration Type ISTO Response Response Ratio Specified Cone an eee eens dee a a de i Baii da KWAO iz hydroco sone met stestosterone 2 steroids03 Standard Method Settings 7031020 2314238 3 038 2 500 progesterone a steroidso4 Standard Method Settings 3666547 2595302 1 413 1 250 4 steroids05 Standard Method Settings 1782495 2403939 0741 0 630 5 steroids13 Standard Method Settings 13449804 2361619 5695 5 000 e steroids14 Standard Method Settings 7160845 2394666 2 990 2 500 steroids15 Standard Method Settings 3574151 2567027 1 392 1 250 8 _ steroids 16 Standard Method Settings 34771261 2530482 13 741 12 000 Selected Chromatogram v gt a ISTD Chromatogram v dda Calibration Curve steroide02 TIC SM T RT 0 30 1 05 NL 9 7166 steroide02 TIC SM T RT 1 63 2 38 NL 3 065 hydrocortisone F Full me2 363 30 40 00 150 00 375 00 F Full m2 303 30 40 00 100 00 310 00 Y 0 0293758 1 14184 R 2 0 9996 W Equal 5 PT 0 68 pea RT 2 00 14 ia eo 12 10 z 604 2 3 2 4 Ee ad g
29. LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View Run Sequence Dialog Box Wii Thermo Scientific The Acquisition view Run Sequence dialog box allows you to select acquisition options and processing actions for the Acquisition Sequence It also allows you to run one sample or a sequence of samples After you have created your Acquisition Sequence click on the Acquire button in the viewbar to open the Acquisition view Run Sequence dialog box See Figure 20 Figure 20 Acquisition view Run Sequence dialog box Run Sequence Acquisition Options User robert h Run Rows 1 13 Comment Instrument Start Instrument T50 Quantum Start When Ready Change Instruments Processing Actions C Process after Acquisition henerate Selected he Instrument Method C Use Startup Method C Use Shutdown Method Programs Pre Acquisition Post Acquisition Run Syncronously C Pre Acquisition _ Post Acquisition After Sequence Set System On O Standby Oot LCquan User Guide 43 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View 44 LCquan User Guide To run a series of samples in the current Acquisition Sequence 1 In the Run Rows text box type the row numbers of the samples that you want to run The rows must be consecuti
30. Method C Xcalibur 28 un2002 WB_28Jun2002 W B_28 e Metutbe Inbar ty AS Status Oven Tray Enor log Target invection Vial postion A 14 Injection volume uL 10 00 AS state Waking for pump Door state Close Maintenance due No For Help press F1 46 LCquan User Guide gt COOSSOCOC Oe nei 216 00 40 00 19350 17450 Number of taces 13 1158 115 9 1164 16 2 1163 1164 10 3 2005 9 57 AI Thermo Scientific 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View The control buttons allow you to modify the acquisition or real time display They are described in Table 3 Table 3 Control buttons in the Acquisition view Status page and their functions Control Button Function D Start Resume Starts an acquisition or restarts an acquisition that has been paused n Pause Pauses the acquisition after the current sample has been completely acquired When clicked on again it restarts the acquisition B Stop Allows you to stop the acquisition of a sample or sequence that belongs to the current workbook You can choose to stop the acquisition of the current sample and pause to stop the acquisition of the current sequence and pause or to stop the acquisition of all sequences a Screen Pause Pauses the chromatogram and spectrum display The acquisition continues When clicked on again it restarts the display 4 Display Spectrum Displa
31. OQAMS MS MS Mode Parent Product Neutral Loss Data Type Centroid Profile Scan Parameters Source CID Scan Range Collision Energy 10 First Mass m z 150 000 2 j Last Mass m z 1500 000 Accurate Mass Mode otf x Scan Time s 1 00 rs Q1 Peak Width FWHM 0 70 v APPI Lamp J Set Mass m z 1000 000 Q3 Peak Width FWHM 0 70 On Oot Collision Energy Vi 10 EnergyRamp evi 0 Micro Scans 1 x Copy ScanE vent Paste ScanE vent Thermo Scientific LCquan User Guide 25 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Instrument Setup View This section contains the following topics e Creating Instrument Methods e Importing an Existing Instrument Method Creating Instrument Methods Although you do not need to create Instrument Methods to follow the quantitative analysis procedure described in this manual the general procedure is outlined below For specific information on how to set up a particular instrument refer to the online Help To create Instrument Methods for your configured instruments 1 Select an instrument by clicking on its icon in the viewbar LCquan displays one or more pages of instrument parameters Note You must configure the instrument before you can define an Instrument Method If you need to configure an instrument you must first close LCqua
32. Status Page 1 ke cece eee 45 Processing Data While Continuing to Acquire Data 0 50 Chapter3 Exploring Your Data 00 cece cece eee eee eee eens 51 Abou the Explore View vata wet cig ee iat ei ee ee ware eats 52 Opening a Raw File and Displaying the Chromatogram 52 Generating a Peak Wisin sti Bas aah lins Boa ores Seana eh what alan 54 Identifying Components he puiiee owt eee ted eere 57 Reviewing the Explore Resultsio ie weeesen eats wenliga wane antes 59 Creating a Quan Component from the Peak List 0 000040 61 Additional Features of the Review and Explore Results Page 62 Sample Selector in reis cuna wy satiate Ms akity apd at aan R tite Seuits 63 LOCKS ee krpata seeks te NEN Ne Ak oh RE ea ie he a A 65 General Parameters Plot ossa vs sess a ern nai a eee eae here a ane abs 66 Status DIOL A cat ee Riese hae tar koe tA ee De a ae hes ies 67 Status REPOT ch a he atti Laat eh neem inant E aE Riese 68 Reyiew IS AVS tos ses wes oige aon Mk dat a aha ee a aha ae Ni Ria i a alt 69 Exporting the Active Raw File to Qual Browser 00000000 70 Chapter4 Creating a Processing Method 0 ec cece ee eee teenies n Opening the Create Method Page and Using the New Method Wizard 72 Specifying the Component Identification and Integration Parameters 75 Identifying the Internal Standard 44 fos n tee ek dae bedded 75 Specifying Calibrat
33. View Width text box e The correct scan filter in the Filter text box is matched to the component If the peak has not been identified properly repeat the procedure outlined in this topic and modify the component identification parameters 5 Enter the settings for hydrocortisone a Select hydrocortisone from the Component list b Enter or select the following settings e Detector MS e Trace Mass Range e Masses 267 2 309 2 e Smoothing 7 e Peak Detection Algorithm Genesis e Filter c Full ms2 363 30 40 150 00 375 00 c Click on Apply to apply the settings LCquan User Guide 79 4 Creating a Processing Method Specifying the Component Identification and Integration Parameters 6 Specify the expected retention time of hydrocortisone a Inspect the mass chromatogram and note the retention time of hydrocortisone In this example the hydrocortisone peak is labeled R7 0 67 b In the Expected text box type 0 67 to specify that the retention time of hydrocortisone is 0 67 min c Select the Adjust Using methyltestosterone check box to use the retention time of methyltestosterone to adjust for the retention time drift of hydrocortisone 7 Integrate the hydrocortisone chromatogram peak a Click on the chromatogram peak in the Chromatogram pane b Click on Apply to integrate the chromatogram peak See Figure 39 Figure 39 Create Method Identify Components page of the Quantitate view showing the integrated ma
34. and enhancement e System instability e Variations in the source conditions For maximum precision the ISTD component should be added as early as possible to the start of the sample workup particularly in those quantitative methods that require sample manipulations such as extraction cleanup and dilution Since the ISTD and non ISTD components are analyzed together the internal standard quantitative analysis approach has the advantage that it corrects for injection and other sample handling errors The ISTD must behave chemically in an identical or similar manner to the target compound through the extraction cleanup and analytical processes The ISTD component can also be added as the last step of sample preparation prior to the sample s use to compensate for fluctuations in the reproducibility of the sample injection In general ISTDs are used in a quantitative analysis experiment as follows 1 A series of standard solutions containing known concentrations of the target compound and ISTD are analyzed Then the ratio of the target compound and the ISTD detector responses are plotted as a function of the corresponding ratio for the two quantities present in the solution 2 A fixed amount of the ISTD is added to each sample prior to any manipulation Then after the samples are prepared and analyzed the quantity of the target compound present in an unknown sample can be obtained from the calibration curve See Figure 3 Ideally a
35. comparing plots 65 Component List 102 components creating from peak list 61 exporting names from peak list 56 naming in Processing Method 75 configuring instruments 26 control buttons and functions Acquisition view 47 copying Acquisition Sequence to Processing Sequence 96 Processing Sequence to Acquisition Sequence 38 Create Method Define Calibration Settings page illustrated 76 Create Method Identify Components page 80 LCquan User Guide 129 Index D Create Sequence Review and Edit page using 97 Create Sequence Select Files page illustrated 93 opening 88 using 89 deoxycorticosterone calibration curve figure 106 creating quan component 61 naming 76 77 peak list illustrated 61 detection limit 6 documentation vii E editing Acquisition Sequence 39 peak detection and integration settings 109 Processing Sequence 97 Excel reports 116 Explore Sequence 63 Explore view additional information 62 audit trail 52 general parameter plots 66 introduction 52 locks 65 review displays 69 sample selector 63 status plots 67 status reports 68 steroids example 52 exporting Acquisition Sequence 42 external standards defined 7 F file and folder structure 16 File Information pane note 91 flow diagrams acquiring data 126 creating study or workbook 124 creating study or workbook Citrix 125 processing data 127 G general parameters plots Explore view 66 Genesis peak detection 77 130 LCquan Use
36. g 507 H EE Tis 2 40 4 2 s 0 0 2 4 6 8 12 os os 086 of oe os 10 Time min Time min ng For Help press F1 Thermo Scientific 10 19 2005 12 42 PI LCquan User Guide 101 6 Processing the Raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Table 7 Sample status and corresponding row color in Result List Sample Status Row Color Peak Not Found Dark Pink Sample Excluded Dark Pink QC Failed Orange Response High Pale Orange Response Low Pale Orange Calculated Amount Is Negative Yellow Calculated Amount is Undefined Orange The Component List contains the components that are defined in the current Processing Method When you select a component in the Component List all of the other page elements are updated to display the results for that component The Calibration Curve pane displays a plot of area ratio or area versus concentration or amount for the selected component The ISTD Chromatogram pane displays a plot of relative intensity versus retention time of the internal standard in the current sample if available Finally the Chromatogram pane displays a plot of relative intensity versus retention time of the selected component in the current sample To review the calibration standards Note The steroids data set does not contain QC samples 1 In the Component List on the right side of the page select the hydrocortisone target compound L
37. how to specify the identification settings for the target compounds and internal standard e Identifying the Internal Standard e Specifying Calibration Curve Parameters for the Target Compounds e Entering the Component Identification Parameters and Integrating the Peaks Identifying the Internal Standard In the last section you specified to import the component names and calibration levels from the Acquisition Sequence into the Processing Method Proceed as follows to identify the internal standard To identify methyltestosterone as the internal standard compound and specify calibration parameters 1 Select methyltestosterone in the Component list 2 Click on the Calibration tab to display the calibration settings 3 Select Component Type ISTD to specify that methyltestosterone is the internal standard 4 Enter 0 63 in the ISTD Amount box and ng in the ISTD Units box to specify that there is 0 63 ng of internal standard Click on Apply to apply the settings If necessary click OK to dismiss the warning Specifying Calibration Curve Parameters for the Target Compounds Thermo Scientific To specify the calibration curve parameters for the target compounds 1 Specify the calibration curve parameters for hydrocortisone a Select hydrocortisone in the Component list b On the Calibration page select or enter the following Component Type Target Compound Target Units ng Calibration Curve Type Linear Re
38. or Last Used eer eae i Workbook LCquan Opens Last Used View I Select Legacy Startup Method Page Instrument Method Page Shutdown Method Page Quantitate View See Quantitate Flow Diagram Click on Method button Create Method Page Click on Sequence button Create Sequence Page Survey Page Click on Survey button Review All Results Page Click on Review All button Review Reports Page Click on Reports button Thermo Scientific A LCquan Flow Diagrams Figure 58 LCquan for Citrix flow diagram showing the steps for creating a new study or workbook and the two main views in dark gray LCquan for Citrix Flow Diagram Open Existing Create New Study or Create New Study or orlLastUsed IT 1 audy and Workbook X Workbook LCquan Opens Workbook Workbook or Open Sette Last Used Vi Names Page Existing Workbook renews Click on Next Import Raw Files Import from Legacy Files Select Legacy Files Page Instrument Method Click on Next Acquisition Sequence j and a Import fron Select Instrument Method optional i Existing Workbook Select Workbook Acquisition Processing Sequence KOERA Parameters cick on Next Page and Processing Method Click on Next and Finish Click on Next Do Not Click on and Finish Import Next amp Prior Data Finish Quantitate View Explore See Quantitate View Flow Diagram
39. properly you are ready to review all of the results e Ifyou want further information about reworking the data refer to the following topics Excluding a Calibration Standard from the Calibration Curve on page 107 Modifying the Peak Detection and Integration Settings on page 109 and Integrating Peaks Manually on page 110 e Ifyou want to review all of the results go to Reviewing All Results on page 112 Thermo Scientific LCquan User Guide 105 6 Processing the Raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs 106 LCquan User Guide Figure 50 Calibration curves for the steroids example Area Ratio Area Ratio Area Ratio hydrocortisone Y 0 000741304 0 554088 K R 2 0 9997 VV Equal 7 ng deoxycorticosterone Y 0 00319805 0 28629 K R 2 0 9992 Wi Equal 30 25 20 00 progesterone Y 0 0126487 0 8629886 X R 2 0 9992 V Equal 9 8 7 Thermo Scientific 6 Processing the Raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Excluding a Calibration Standard from the Calibration Curve LCquan allows you to exclude specific calibration standards from the calibration curve Of course you should follow the procedures of your laboratory for proper treatment of calibration data You can exclude calibration data in the following ways By using the Exclude column of the Result List Ensure that the S
40. rather than a precise one This form of measurement is generally called semi quantitative analysis This chapter discusses quantitative analysis in detail Quantitative analysis consists of the following steps e Preparing samples e Developing a suitable chromatographic method e Calibrating the mass spectrometer s response e Analyzing the samples Reviewing the results It is beyond the scope of this manual to describe sample preparation and chromatographic method development This manual assumes that you have achieved these important prerequisites to high quality quantitative analysis Refer to the Getting Started and Hardware manuals for your autosampler LC pump or MS pump and mass spectrometer for guidance in these areas 2 LCquan User Guide Thermo Scientific 1 Introduction Considering the Variables of Quantitative Analysis by LC MS MS Considering the Variables of Quantitative Analysis by LC MS MS The combination of liquid chromatography LC and mass spectroscopy MS sets up a unique set of considerations Chemists who are familiar with quantitative analysis using one or the other of these analytical tools can find the information in the following topics useful e Using LC for Analyte Separation e Using MS MS for Analyte Detection Using LC for Analyte Separation Thermo Scientific Chemists who work with LC are accustomed to developing methods for their analytes of interest that take into account a variety of parameter
41. steroids15 Gd steroids16 Gd steroids17 Field value For Help press F1 _ NOT SAVED 10 17 2005 12 53 PM 3 Associate samplea sampleb samplec sampled and samplee with the unknown sample type a Click on the Unknowns tab in the Selected Files pane b In the Available Files pane click on samplea to select the file c Hold down SHIFT and click on samplee d Click on Add LCquan adds the five selected raw files to the Unknowns page of the Selected Files pane If necessary delete the blank line above samplea by selecting it and then clicking Remove 92 LCquan User Guide Thermo Scientific 5 Creating a Processing Sequence Associating Raw Files with Sample Types The Create Sequence Select Files page should look like the one shown in Figure 45 Note The steroids data set does not contain any quality control or blank samples When a data set does contain QCs or blanks you associate the raw data files to the QC or blank sample type in the same manner as above Figure 45 Create Sequence Select Files page of the Quantitate view showing the proper association of raw files to the unknown sample type for the steroids example ON L quan Steroids Workbook EEK File Zoom View Apps Options Help y PaHE ELE TWA COCOSSOCO i O 0 Create Sequence select files review and edit pee e Avalable kies Standards QCs Blanks Unknow
42. to Importing the Components and Levels into the Acquisition Sequence on page 84 e The steroids data set does not contain any quality control or blank samples When a data set does contain QCs or blanks you associate the raw data files to the QC or blank sample type in the same manner as above 9 Specify that there are no QCs a Add QCs b Click on Next You can use the QC levels from the Processing Method if one exists in the workbook or you can have LCquan generate names for the levels by appending numerical values to a base name 10 Specify temporary component names for the steroids e Number of Components 4 e Component Base Name Component Click on Next 11 Accept the default labels and the values for the five user defined text boxes that appear in the Sequence Header area of the page The information that you enter in these text boxes appears in the header of the raw file Click on Next 32 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View 12 Click on Finish to close the wizard LCquan displays the Acquisition view Setup Sequence page See Figure 15 Figure 15 Acquisition view Setup Sequence page Cquan Steroids Workbo Options Help _Ele Change Actions Yiew Apps 08H Gy W steroids001 g 5 E 2 Study Client Laboratory Company Phone 1 gt OOOO00
43. 0 Time min LCquan User Guide 67 3 Exploring Your Data Additional Features of the Review and Explore Results Page 68 Status Report LCquan User Guide A status report is a list of status information for all parameters at one retention time See Figure 34 Use the arrows to move between time points of the chromatogram Note that status information is acquired less frequently than the chromatogram so the status information does not always change Figure 34 Status report device MS a a Status Report API SOURCE Source Yoltage VY Source Current uA Vaporizer Thermocouple OK Vaporizer Temp CX Sheath Gas Flow Rate Aux Gas Flow Rate Capillary RTD OK Capillary Voltage V Capillary Temp C 8 kV supply at limit VACUUM Vacuum OK lon Gauge Pressure OK lon Gauge Status lon Gauge x10e 5 Torr Convectron Pressure OK Convectron Gauge Torr RT 2 011 Thermo Scientific 3 Exploring Your Data Additional Features of the Review and Explore Results Page Review Displays You can customize the layout of the Review and Explore Results page The five standard layouts are described in Table 5 Use the Current Review Display list to select a layout Table 5 Review displays and their layouts Display Layout Multi Peak View On the left side of the page are two panes defaulted to Chromatogram and Spectrum On the right s
44. 0 00 Example Study 76 Prev Row Row 1 Next Row Ceara Compress l Help R Acquisition Sequence History Sequence information For multiple seque ___ __samptetype__ _Filetame Levet Cal Fie Sample Sth Inst M steroids002 cal02 C Xcalibur QuanRoot Example Study C Xcalibur QuanRoo 3 3 Standard steroids003 cal03 C XcaliburQuanRootExample Study C XcaliburQuanRoo 4 Standard steroids004 cal04 C XcaliburiQuanRootlExample Study C XcaliburQuanRoo 5 Standard steroids005 cal05 C XcaliburiQuanRootlExample Study C Xcalibur QuanRoo je Unknown steroids006 NA sample006 C XcaliburiQuanRoot Example Study C Xcalibur QuanRoo Unknown steroidsO07 NA sample007 C XcaliburiQuanRoot Example Study C XcaliburQuanRoo ja Unknown steroids008 NA sample008 C XcaliburiQuanRoot Example Study caliburiQuanRoo a Unknown steroids009 INA sample009 C XcaliburiQuanRoot Example Study C XcaliburQuanRoo 10 Unknown steroids010 NA sample010 C Xcalibur QuanRootExample Study C Xcalibur QuanRoo Standard steroids011 calit C XcaliburiQuanRootlExample Study C Xcalibur QuanRoo 12 Standard cal02 C XcaliburiQuanRootlExample Study C Xcalibur QuanRoo cal03 C XcaliburQuanRootiExample Stud wanRoo i e calg C Xcalibur QuanRootExample Stud uanRoo steroids015 cal05 C Xcali
45. 00 F c Full ms2 363 30 40 00 150 00 375 00 349 54 1005 1005 RT 0 68 x 2 S 6 6 o a amp Relative Intensity a 3 Relative Intensity yo Ss S E sali poitiiitiiiitiis silir 0 88 0 93 03 0 4 os 06 07 08 0 9 10 Time min 5 For Help press F1 NOT SAVED 10 3 2005 1 33 PM 80 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Specifying the Component Identification and Integration Parameters 8 Enter the settings for deoxycorticosterone a Select deoxycorticosterone from the Component list b Enter or select the following settings e Detector MS e Trace Mass Range e Masses 109 0 253 1 295 2 e Smoothing 7 e Peak Detection Algorithm Genesis e Filter c Full ms2 331 30 40 100 00 350 00 c Click on Apply to apply the settings 9 Specify the expected retention time of deoxycorticosterone a Inspect the mass chromatogram and note the retention time of deoxycorticosterone In this example the deoxycorticosterone peak is labeled R7 1 39 b In the Expected text box type 1 39 to specify that the retention time of deoxycorticosterone is 1 39 min c Select the Adjust Using methyltestosterone check box to use the retention time of methyltestosterone to adjust for the retention time drift of deoxycorticosterone 10 Integrate the deoxycorticosterone chromatogram peak a Click on the chromatogram peak in the Chrom
46. 2 363 30 150 00 375 00 v _ Constrain Peak Width pom ee Retention Time Peak Ht Expected min 0 68 Window sec 30 0 Expected min dow sec o a Window Use as RT Ref View Width min 1 00 Relative Qualifier ion coelution Adjust Using methyltestosterone O Absolute 0 100 min Apply Cancel Save As Default Help e Add le Delete O Rename J IRC Chromatogram v dra Spectrum bs 7 10 327 10 SM T RT 0 46 1 00 NL 5 01E5 steroidz02 miz 267 20 267 20 303 20 303 20 SM 7 RT 0 46 1 00 steroids02 94 RT 1 01 NL 2 08E3 s Fen maa 389 90 150 00 375 001 F Full ms2 363 30 150 00 375 00 F c Full ms2 363 30 40 00 150 00 375 00 ose 303 16 1005 a 100 35 4 30 4 90 85 80 4 80 15 4 704 70 654 ih 604 2 60 4 2 504 o 50 2 oe 2 a0 404 g 30 20 10 05 06 os Time min Time min For Help press F1 Thermo Scientific NOT SAVED 10 24 2005 4 36 PI LCquan User Guide 83 4 Creating a Processing Method Importing the Components and Levels into the Acquisition Sequence Importing the Components and Levels into the Acquisition Sequence In the topic Opening the Create Method Page and Using the New Method Wizard on page 72 you imported the component names and calibration levels from the Acquisition Sequence into the Processing Method You can also do the reverse that is import the component names and calibration levels from the Processing M
47. 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View Assigning Calibration or OC Levels You can define or modify the calibration levels and QC levels that are specified in the Acquisition Sequence by using the Acquisition Levels dialog box To open this dialog box right click in the Sequence table and choose Standard and QC Levels from the shortcut menu See Figure 20 Figure 19 Acquisition Levels dialog box Acquisition Levels Components Levels component attevet ca ComponentO2 ComponentO3 4 Component04 OC Levei amount wrest The Acquisition Levels dialog box contains the Components table and the Levels tables You can enter the names of the components present in your sample in the Component table and then define calibration or QC levels for each component If you have already defined components in the Quantitate view you can import them into the Processing table by clicking the Import Processing Components button Any calibration or QC levels that you entered into the Acquisition Sequence appear in the Levels tables You can define the amount for each calibration level and the amount and test the acceptable difference between the known and measured amount for each QC level You can also create and define new levels If you want to load the set of default levels click on the Load Default Levels button Thermo Scientific LCquan User Guide 41 2 Preparing to Perform Qua
48. 35 100 90 Relative Intensity 3 dda Spectrum steroids04 1 RT 0 01 NL 4 94E2 ae 247 48 150 200 250 300 350 miz For Help press F1 Thermo Scientific _ NOT SAVED 10 7 2005 9 57 AM LCquan User Guide 53 3 Exploring Your Data Generating a Peak List Generating a Peak List LCquan can generate a peak list A peak list is a table of peak properties such as retention times and peak areas of the major peaks in the chromatogram You can use the peak list to identify and name quan components and then export the component names and properties to the Quantitate view See the topic Creating a Quan Component from the Peak List on page 61 To create a peak list corresponding to the four major peaks of the steroids example 1 Select Peak List in the right side pane 2 Specify the peak list parameters e Select Top Peaks Enabled e Select by Area e Num to Select 4 3 Click on Apply 4 LCquan generates a peak list for the four major peaks in the chromatogram See Figure 25 Note In Explore only the peak name is editable in the peak list You can have LCquan generate names for the peaks by right clicking on the peak list and then choosing Create Peak Names gt For All Peaks in List from the shortcut menu LCquan generates peak names in the form Peak x xxxx where x xxxx is the retention time at the peak apex The component name is the same as the peak name See Fig
49. 43 351 14 895 20 i i j BE 6 704 F E g i ae a ji 3 4 no so E il 5 h2 amp 130 w w i oa w 18 h 22m H 20 i EI l 2a 4 220 4 i 24 M4 For Help press F1 Thermo Scientific NOT SAVED 11 17 2005 2 28 PI LCquan User Guide 55 3 Exploring Your Data Generating a Peak List Figure 26 Peak list with auto generated names Peak List j1 Peak 0 675 3621463 100 000 2715440 74 982 2633421 72 717 1569926 43 351 You can change peak names by selecting the current name and then typing a new name or by using the Peak Names List Editor To open the Peak Names List Editor right click on the peak list and choose Modify Peak Names from the shortcut menu The Peak Names List Editor also allows you to import and export peak lists Once you name the components you can export the component names and settings to the Quantitate view by right clicking on the peak list and then choosing Create Quan Components gt For All Peaks in List from the shortcut menu See the topic Creating a Quan Component from the Peak List on page 61 Go on to the next topic Identifying Components 56 LCquan User Guide Thermo Scientific Identifying Components 3 Exploring Your Data Identifying Components When LCquan acquires data it creates unique scan filters according to the type of experiment you specify in the Instrument Method When you load a raw file LCquan lists in th
50. 68 AT 2 00 1 100 14 30 PA 12 t 10 A 2 2 Ai x x 50 j E a Ri 4 2 10 0 i Tr 0 2 4 6 8 10 12 o4 Os 06 or 08 0 3 10 Ww 18 13 20 21 22 23 Time min Time min ng deoxycorticosterone methyltestosterone progesterone For Help press F1 108 LCquan User Guide Heather Robert w NOT SAVED 10 19 2005 2 33 PI Thermo Scientific 6 Processing the Raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Modifying the Peak Detection and Integration Settings You can modify the peak detection and integration settings from the Survey view or the Review All view To modify the peak detection and integration settings 1 Select a sample by clicking on the appropriate row in the Result List 2 Right click in the Chromatogram pane to display the shortcut menu 3 From the shortcut menu choose User Peak Detection Settings to open the User Identification Settings dialog box 4 To modify the peak detection settings click on the Detection tab and change the settings as necessary 5 To modify the peak integration settings click on the Integration tab Then change the settings as necessary to correct problems with noise in the peak unresolved peaks or peak tailing 6 To modify the advanced peak detection parameters click on the Advanced tab Then adjust the settings as necessary to improve peak detection and integration if baseline noise
51. 84 X R 2 0 9996 V Equal acl hydrocortisone For Help press F1 LCquan User Guide NOT SAVED 10 19 2005 3 45 PI Thermo Scientific 1 Introduction Introduction to Quantitative Analysis with LCquan LCquan uses a Processing Method to automatically determine the concentration or amount of the sample being analyzed The Processing Method measures the response of the detection system to the compounds in your sample by integrating the area under each peak After several known standards are measured LCquan constructs a calibration curve which it uses to accomplish the quantitative analysis of unknown samples LCquan provides a number of options for fitting the calibration data to generate the calibration curve You can have LCquan fit the calibration data to the following curve types e Linear e Quadratic e Linear log log e Quadratic log log e Average response factor RF e Point to point e Cubic spline e Locally weighted You can also have LCquan weight the calibration data with the following weighting functions when performing the least squares fit to the calibration data e Equal e 1 X e 1 X e 1 Y e 1 Y 1 S2 where S2 X Y2 In addition you can have LCquan ignore the origin use the origin as a data point or force the calibration curve to include the origin The Quantitate view contains the following pages Create Method Create Sequence Survey Review All and Review Reports
52. 87 7 Processing the Raw Files on page 100 8 Reviewing the Calibration Curve and QCs on page 100 9 Reviewing All Results on page 112 Thermo Scientific 1 Introduction LCquan for Citrix Note Sections of this manual that provide only additional information and are not necessary for the steroids quantitative analysis are clearly marked You can skip these sections while performing the analysis however they provide important information and should be read at a later time LCquan for Citrix LCquan supports Citrix Presentation Server Citrix Presentation Server is used for application virtualization to manage the LCquan software configuration and maintenance The LCquan virtual application runs on centralized servers only keystrokes and mouse clicks travel over the network from the user s computer and only screen images travel over the network to the user s computer You need to validate LCquan only on the server LCquan 2 5 SP1 and later versions support the Citrix environment for data processing LCquan for Citrix includes only the Explore and Quantitate views of LCquan Certain actions within LCquan such as data acquisition can be done only on a local validated computer and not the server Thermo Scientific LCquan User Guide 19 Preparing to Perform Quantitative Analysis of Steroids Data This chapter describes how to start LCquan and create a new study and workbook in preparation for analyzing pre ex
53. Cquan automatically updates the Result List the Chromatogram and ISTD Chromatogram panes and the Calibration Curve pane 2 Ensure that the calibration standards are displayed in the Result List If necessary click on the Standards tab at the bottom of the Result List to display the calibration standards 3 In the Calibration Curve pane inspect the calibration curve at all concentrations When you process your own data evaluate the calibration curve according to the criteria used in your laboratory 4 Inspect the calibration curve at low concentrations a Click and drag the mouse diagonally across the low concentration data points in the calibration curve See Figure 48 b Release the mouse button LCquan displays only the selected data points See Figure 49 102 LCquan User Guide Thermo Scientific 6 Processing the Raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Figure 48 Calibration curve showing the selection of the low concentration data points hydrocortisone Y 0 0293758 1 14184 K R 2 0 9996 VV Equal 2 T oe G D q Figure 49 Calibration curve showing only the low concentration data points hydrocortisone Y 0 0293758 1 14184 K R 2 0 9996 Vi Equal 40 3 5 3 0 25 2 0 Area Ratio 15 1 0 05 0 0 5 Reset the scaling of the X and Y axes of the calibration curve to full scale by clicking on the Reset Scaling button Tip You can change the
54. Display Settings dialog box showing the settings for five user defined traces Realtime Display Settings Options 5 Max number of visible chromatograms _ Link chromatogram time ranges Show user defined traces C Show spectrum trace C Use user defined time range User defined traces Type Scan filter TIC M TIC c Full ms2 303 30 40 100 00 310 00 v c Full ms2 315 30 40 100 00 320 00 v c Full ms2 331 30 40 100 00 350 00 v c Full ms2 363 30 40 00 150 00 375 Trace properties Title Trace type TIC Scan filter c Full ms2 363 30 40 150 00 375 00 Range s Smoothing points 1 Using the Spectrum Pane The Spectrum pane displays the data for the currently selected scan in the currently selected chromatogram Select a scan by left clicking on a chromatogram A red vertical line appears at the scan you selected in the Chromatogram pane and the spectrum that corresponds to that scan appears in the Spectrum pane See Figure 23 You can choose to apply a scan filter to the data based on the selected chromatogram Right click in the Spectrum pane and choose Filter Spectrum from the shortcut menu A check mark appears next to the command To remove the filter and show every scan choose this command again Thermo Scientific LCquan User Guide 49 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View You can use the dis
55. FWHM 0 70 n Oot Collision Energy WV 110 Energy Ramp ev 0 Menica 1 Copy ScanE vent Paste ScanE vent Thermo Scientific LCquan User Guide 11 1 Introduction Introduction to Quantitative Analysis with LCquan 12 Acquisition View Figure 5 AN LEquan ile Change Actions View Options Help The Acquisition view of LCquan see Figure 5 allows you to set up a sequence of samples for acquisition The sequence can contain unknown samples calibration standard samples quality control samples and blank samples Once you have defined a sequence you can specify acquisition options and processing action options for the sequence and then start running it The Acquisition view also contains a real time display so that you can watch updates of the acquisition status and the data being acquired The Acquisition view contains the Sequence Setup page the Run Sequence dialog box and the Status page Note The Acquisition view is not available in LCquan for Citrix Acquisition view Setup Sequence page Steroids Workbook Apps Acquisition Comment FileName Sample Name Sample02 Prev Row Row Next Row Samplet3 steroids Client Laboratory Company Name Phone steroids002 EL u WR aooo Acquisition Sequence History Example Study C Xcalibur QuanRootExamp
56. Help gaug casn Current Review Display Quantitate Rawtile steroids04 raw J amp amp Explore Sequences Quantitate Sequence i W Processing Sequence Acquisition Sequence History w AcqSequence 1 Load 1 Remove Rename gt a LS 2j0O00S0001 00 Chromatogram Relative Intensity x 3 a So e a Ss 8 wenn poirtiiitiris a T gt n s im o Piositiiitis steroids04 TIC SM 7 RT 0 01 3 79 NL 9 59E5 RT 0 68 RT 1 99 RT 3 15 Time min doa Spectrum Relative Intensity 1005 steroids04 59 RT 0 68 NL 1 15E5 F c Full ms2 363 30 40 00 150 00 375 00 309 16 steroideO TIC SM 7 RT 0 49 0 95 NL 9 5965 Retatue heast Retate heist steroide steroide 1005 g 8 Tus 1005 3 mn 04 TIC SM 7 RT 0 90 2 09 NL 9 5965 04 TIC SM 7 PT 2 70 3 83 NL 3 53E5 Multi Peak Plot RT 0 68 oT Time min 24 Time min PT 3 15 Time min For Help press F1 NOT SAVED 10 7 2005 10 12 AM Thermo Scientific LCquan User Guide 59 3 Exploring Your Data Reviewing the Explore Results 5 Click on the second chromatogram
57. Quantitate i gt Install Logs epi methods ii QuanRoot call2 E Example Study cal03 S Practice Study call B Steroids Workbook cal05 0 Exports amp Imports a comia a Temp B A system x Ea Add gt gt gt gt samplel Nella kc Remove Remove LE samplec steroidsO2 ter steroids03 secs GJ steroids05 Gad steroids13 Q steroids14 A steroids15 E steroids16 E steroids17 Field Value For Help press F1 _ NOT SAVED 10 17 2005 12 48 PI 90 LCquan User Guide Thermo Scientific 5 Creating a Processing Sequence Associating Raw Files with Sample Types 2 Associate the calibration data files with the correct calibration levels a Thermo Scientific Ensure that the Standards page of the Selected Files pane is displayed If the Standards page is not displayed click on the Standards tab to display it In the Standards page click on cal01 to select the first calibration level In the Data Files pane click on steroids05 to select the file Click on the Add button LCquan adds the selected raw file to the cal01 calibration level in the Selected Files pane Tip You can also associate a raw file with a sample type by clicking on the raw file in the Data Files pane and then dragging it to the appropriate level or page in the Selected Files pane Associate the raw files and calibration levels as shown in Table 6 The Create Sequence Select Files page should look like the one shown in Figure 45 Tabl
58. This chapter describes how to process the raw steroids data by using the Processing Method and the Processing Sequence that you created in the previous chapters It also explains how to review the results for each component in each raw file and how to produce reports Contents e Processing the Raw Files e Reviewing the Calibration Curve and QCs e Reviewing All Results e Creating and Reviewing Reports e Locking the Workbook e Saving the Workbook and Exiting LCquan Thermo Scientific LCquan User Guide 99 6 Processing the Raw Files and Reviewing the Analytical Results Processing the Raw Files 100 Processing the Raw Files LCquan processes the raw files in the Processing Sequence when you open the Survey page the Review All Results page or the Review Reports page of the Quantitate view LCquan uses the peak detection and integration parameters in the Processing Method to detect and integrate peaks for all components It then uses the calibration parameters in the Processing Method to build a calibration curve with which it quantitates the QCs and unknowns Note You need to define a Processing Method and a Processing Sequence before you can open the Survey page the Review All Results page or the Review Reports page Although you can review the results in any order in this example you first review the calibration standards Reviewing the Calibration Curve and OCs You use the Survey page of the Quantitate view to review and rewo
59. Update the Processing Sequence When Acquiring check box to update the sequence If you are in the Quantitate view and currently acquiring data when you select this option a message box prompts you to update the current Processing Sequence with the most recent list of samples acquired If you do not respond to the message box after 5 s LCquan automatically updates the sequence 50 LCquan User Guide Thermo Scientific Exploring Your Data Thermo Scientific This chapter describes the features of the Explore view You can use the Explore view to display a multi component chromatogram and then to experiment with peak detection and integration parameters to see how they affect the chromatogram Contents About the Explore View Opening a Raw File and Displaying the Chromatogram Generating a Peak List Identifying Components Reviewing the Explore Results Creating a Quan Component from the Peak List Additional Features of the Review and Explore Results Page LCquan User Guide 51 3 Exploring Your Data About the Explore View About the Explore View The Explore view allows you to display a multi component chromatogram and then to experiment with peak detection and integration parameters to see how they affect the chromatogram In the Explore view you can also create and export quan components to the Processing Method to get it started In Explore it s expected that you ll be looking at the entire chromatogram and find multiple peak
60. acteristics of your analyte should influence your decisions about the optimum pH of your solvent system and about which probe to use at the interface of the LC and the mass spectrometer With the TSQ Quantum series LTQ series or LCQ series instruments you can use either the H ESI probe the ESI probe the APCI probe or the APCI APPI probe at the interface of the LC The choice of probe depends on the class of compound that you want to analyze and to a small extent on the flow rate of your experiment In general the APCI and the APCI APPI probes accommodate higher flow rates and molecules of a less polar nature than does the ESI probe Refer to the Introduction chapter in the Getting Started manual for your mass spectrometer for a discussion of factors that can influence your decision about flow rates and about which probe to use for optimum transfer of ions into the mass spectrometer Thermo Scientific 1 Introduction Quantitative Analysis Techniques After the ions are inside the mass spectrometer and the parent ions are isolated collision energy is applied to dissociate the parent ions into product ions MS MS The relative collision energy for a particular analysis depends on the structure of the compound that you are analyzing For this reason you optimize the collision energy on your analyte of interest in a tuning experiment Refer to the Getting Started manual for your mass spectrometer for the tuning procedure that describes ho
61. alyze and graph the data by using all of the functions and features of Microsoft Excel Long Excel Summary Displays the Result List of the Quantitate view Processing Sequence and component identification and calibration information in a Microsoft Excel spreadsheet Each component is placed on a separate page worksheet in the main workbook You can review analyze and graph the data by using all of the functions and features of Microsoft Excel If your administrator has set up secure reporting the Create XReport Templates button is unavailable and only the templates in the secure template folder are available The only output option is Save Only which saves the file in pdf format 120 LCquan User Guide Thermo Scientific 6 Processing the Raw Files and Reviewing the Analytical Results Locking the Workbook Locking the Workbook You can create a locked version of a workbook at any time except when data is being acquired A locked workbook is essentially a copy of the workbook and its associated files that cannot be overwritten You cannot save any changes made to a locked workbook and you cannot acquire data in a locked workbook You can create new reports but LCquan does not save the report selections To lock the current workbook Choose File gt Create Locked Version of Workbook LCquan creates a copy of the workbook and its associated files under the current study folder The locked workbook is renamed by appending
62. arameters calibration curves calibration and QC levels and amounts and Result List information for example peak areas and calculated amounts for all components in all samples Ion Ratio Conformation Results Report LCquanlonRatioConformationGraphical xrt Provides the chromatogram traces of the conformation ion and qualifier ions for each component The report also lists sample information peak status and calculated concentrations for each sample Peak Integration Report LCquanPeakIntegration xrt Provides the Results List information for example peak areas and calculated amounts for all components the sample information and the Instrument Method name and Processing Method name if applicable The report includes plots of the chromatogram peaks for all components Processing Method Report Avalon Genesis or ICIS algorithm LCquanProcessingMethod xrt Lists all the peak detection and integration parameters in the Processing Method for each component The report also lists level amounts IRC data and the mass to charge ratios and intensities of the peaks in the mass spectrum for each component Quan Components Peak Results Report LCquanQuanPeakResults_ESTD xrt LCquanQuanPeakResults_ISTD xrt Lists the sample information acquisition conditions and quantitative analysis results for a single sample The report also displays the integrated chromatograms of each target compound and the standard as well
63. ars to indicate the new position of the row in the table The row will be pasted into the table row below the red line Release the mouse button to paste the row into the new position If pasting the row will overwrite an existing row a message box appears to warn you Click on Yes or No as appropriate To remove empty rows from the Acquisition Sequence click on Compress Changing the User Labels and Values You can change the labels and the values for the five user defined text boxes that appear in the Sequence Header area of the page The information that you enter in these text boxes appears in the header of the raw file Right click in the Sequence table and choose User Labels and Values from the shortcut menu to display the Change Sequence User Labels dialog box In the User Label text boxes type the label for each entry In the User Values text boxes either type the text to appear in each box or select a macro name from the drop down list When you select a macro name LCquan displays the actual text that corresponds to the macro name in the Sequence Header For example if you select Workbook LCquan displays the name of the current workbook in the field in the Sequence Header Any changes that you make to the user labels or values with the Change Sequence User Labels dialog box are applied to all rows in the Acquisition Sequence Any new rows that you create use the same labels and values 40 LCquan User Guide Thermo Scientific
64. as the calibration curves Quantify Component Report LCquanQuantifyComponentReport xrt Provides a summary by component of the samples for each component The report includes sample information and quantitative analysis results for example peak areas response and calculated amount and it displays the integrated chromatogram peaks Quantify Sample Report LCquanQuantifySampleReport xrt Provides a summary by sample of the components for each sample The report includes sample information and quantitative analysis results for example peak areas response and calculated amount and it displays the integrated chromatogram peak Quan Results Report LCquanQuanResults xrt Thermo Scientific Provides quantitative results for a single sample LCquan User Guide 119 6 Processing the Raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Table 8 Report options available in Review Reports page Sheet 2 of 2 Report Options Description Excel Report Options Quan Result Grid Generates an Excel report using the current Quan Results column arrangement as specified on the Survey and Review pages of the Quantitate section This column arrangement cannot be modified from the Review Reports page Short Excel Summary Displays the Result List of the Quantitate view in a Microsoft Excel spreadsheet Each component is placed on a separate page worksheet in the main workbook You can review an
65. ating Peaks Manually on page 110 5 In the Component List select a target compound 6 Repeat steps step 3 and step 4 above for each target compound Note For the file samplec target compound ydrocortisone its row in the Result List is shaded pale orange and the Peak Status column displays Response High This means that the response for the sample is greater than the response for any of the calibration standards 7 When you are satisfied that LCquan has detected and integrated the peaks properly inspect the Result List again The calculated amount of steroids in each unknown sample is displayed in the Calculated Amount column of the list See Figure 54 8 Left click in the Chromatogram pane to select it Then right click and choose Show Peak Info from the shortcut menu The Peak Information dialog box opens and allows you to review the peak properties for the selected steroids sample See Figure 55 With the Peak Information dialog box still open select the other steroids files in the Result List and view the peak properties for them For more information about the peak properties refer to the online Help 114 LCquan User Guide Thermo Scientific 6 Processing the Raw Files and Reviewing the Analytical Results Reviewing All Results Figure 54 Review All Results page of the Quantitate view showing the results for the hydrocortisone unknowns Calculated Amount
66. atogram pane b Click on Apply to integrate the chromatogram peak 11 Enter the settings for progesterone a Select progesterone from the Component list b Enter or select the following settings e Detector MS e Trace Mass Range e Masses 255 0 279 2 297 1 e Smoothing 7 e Peak Detection Algorithm Genesis e Filter c Full ms2 315 30 40 100 00 320 00 c Click on Apply to apply the settings Thermo Scientific LCquan User Guide 81 4 Creating a Processing Method Specifying the Component Identification and Integration Parameters 12 Specify the expected retention time of progesterone a Inspect the mass chromatogram and note the retention time of progesterone In this example the progesterone peak is labeled RT 3 16 b In the Expected text box type 3 16 to specify that the retention time of progesterone is 3 16 min c Select the Adjust Using methyltestosterone check box to use the retention time of methyltestosterone to adjust for the retention time drift of progesterone 13 Integrate the progesterone chromatogram peak a Click on the chromatogram peak in the Chromatogram pane b Click on Apply to integrate the chromatogram peak Note The ICIS peak detection algorithm has been designed for MS data and has superior peak detection efficiency at low MS signal levels The Genesis peak detection algorithm has been provided for backwards compatibility with Xcalibur 1 0 studies The Avalon peak detection algorithm
67. bur QuanRootExample Study C Xcalibur QuanRoo EE 2 Ey 2 v ii For Help press F1 Thermo Scientific E NOT SAVED 10 26 2005 12 52 PM PRSLA AL LCquan User Guide 33 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View Editing the Acquisition Sequence for the Steroids Example Although you are not acquiring data in this example in this section you edit the Acquisition Sequence as if you were This Acquisition Sequence would produce the raw files in the steroids data set Later you can use this Acquisition Sequence as the Processing Sequence To edit the Acquisition Sequence 1 Delete rows 1 and 15 a b e Select row 1 by clicking on the 1 at the left of the row Delete row 1 by right clicking on row 1 and then selecting Delete Rows from the shortcut menu Select row 15 by clicking on the 15 at the left of the row Delete row 15 by right clicking on row 15 and then selecting Delete Rows from the shortcut menu Em Click on Compress to remove blank rows 2 Change the file names and calibration levels as shown in Table 1 You can change a file name or calibration level by clicking in the box and then using the down arrow Table 1 Sample types file names and calibration levels in the Acquisition Sequence for the steroids example
68. cal Results Reviewing the Calibration Curve and OCs Figure 51 Survey page of the Quantitate view showing that the last calibration standard in the Result List has been excluded from the calibration curve AM LC quan Steroids Workbook Eile Zoom View Apps Options Help Daug reen Acquisition Explore Quantitate EL UR COOOSSSO n kO Survey review standards and Qs FileName Sample Type Sample Name Integration Type Response ISTD Response Response Ratio Specified Conc 7 2 steroids03 Standard Method Settings 7031020 2314238 3 038 2 500 3 steroids04 Standard Method Settings 3666547 2595302 1 413 1 250 4 steroids05 Standard Method Settings 1782495 2403939 O74 0 630 5 steroids13 Standard Method Settings 13449804 2361619 5 695 5 000 6 steroids14 Standard Method Settings 7160845 2394666 2 990 2 500 7 steroids15 Standard Method Settings 3574151 2567027 1 392 1 250 8 z m a 4 gt AN Standards OCs alka il E 4 gt amp Selected y Chromatogram M gt a isto y Chromatogram dra Calibration Curve steroide02 TIC SM 7 RT 0 30 1 05 NL 3 T1E6 steroids02 TIC SM 7 RT 1 63 2 38 NL 3 30E5 hydrocortisone F c Full ms 363 30 40 00 150 00 375 00 F c Full me 303 30 40 00 100 00 310 00 Y 0 03866409 1381 X R 2 0 9984 W Equal AT 0
69. calibration curve parameters including curve type and level information by using the Calibration Settings dialog box To open the Calibration Settings dialog box right click in the Calibration Curve pane to display a shortcut menu Then choose Calibration Settings from the shortcut menu Thermo Scientific LCquan User Guide 103 6 Processing the Raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs 104 LCquan User Guide 6 Inspect the Result List Check the entries in the Result List for peak detection and integration problems Use the scroll bar at the bottom of the list to see all of the entries b Ensure that the data files correspond to the correct levels and sample types 7 Select the first data file by clicking on the first row of the Result List 8 Inspect the target compounds and internal standard peaks Inspect the component peak in the Chromatogram pane i Ensure that LCquan found the peak LCquan shades the peak it finds gray and marks the starting and ending points of the peak with integration markers ii Ensure that the shaded area accurately represents the contribution of the component to the chromatogram Inspect the internal standard peak in the ISTD Chromatogram pane i Ensure that LCquan found the peak LCquan shades the peak it finds gray and marks the starting and ending points of the peak with integration markers ii Ensure that the shaded area accurately repr
70. click the row number Right click the sequence grid and choose Delete Selected Samples from the shortcut menu e Adding raw files to the Processing Sequence Select the row in the sequence above which you want the new row sample to be located Right click the sequence and then choose Add Sample from the shortcut menu In the Select Rawfile dialog box select the files you want to add and then click Open LCquan obtains the sample information from the selected files and adds the samples to the sequence e Changing the sample type Click the Sample Type box to display the arrow and then select the new sample type from the list e Changing a range of cells You can fill selected columns of selected rows with duplicate text entries or appropriately sequenced number entries Select the row that you want to copy right click the sequence grid and then choose Fill Down from the shortcut menu In the Sequence Fill Down dialog box specify the columns to be included and the range of rows e Modifying the arrangement of the Processing Sequence You can modify the arrangement of the sequence by using shortcut menu commands For example you can change the sorting order of the sequence Right click in the sequence to display the shortcut menu Refer to the online Help for additional information about editing the Processing Sequence Thermo Scientific LCquan User Guide 97 Processing the Raw Files and Reviewing the Analytical Results
71. component in the Explore view and then export it to the Quantitate view Creating quan components is the first step in creating a Processing Method The components that LCquan creates out of the peak list are based on the Explore Method that you define in the Create Explore Methods page In the previous topic you set up a method that s a TIC no filter For steroids this resulted in a chromatogram and peak list with four peaks In general you want to filter first to get one peak in the peak list and then generate its quan component You then add quan components to the Processing Method one at a time To create the deoxycorticosterone quan component 1 Click on the Method button to display the Create Explore Methods page o 2 Enter or select the following Explore Method settings e Detector MS e Trace Mass Range e Masses 109 0 253 1 295 2 e Smoothing 7 e Peak Detection Algorithm Genesis e Filter c Full ms2 331 30 40 100 00 350 00 3 Change the bottom right pane to Peak List 4 Click on Apply to apply the settings See Figure 29 5 Select Peak Name 1 383 and type in deoxycorticosterone to rename the peak See Figure 30 Figure 29 Initial peak list that was obtained with the Explore Method of step 2 Peak List Area of Largest Area of Total Area 258693 100 000 100 000 Thermo Scientific LCquan User Guide 61 3 Exploring Your Data Additional Features of the Review and Explore Resul
72. contains hydrocortisone deoxycorticosterone and progesterone as the target compounds A fourth steroid methyltestosterone is used as an internal standard ISTD The amount of the internal standard in the calibration standards in this example is 0 63 ng Calibration standards were prepared to give five calibration levels with amounts of 0 63 1 25 2 5 5 0 and 10 0 ng This example also includes five unknown samples No QC samples are included This manual leads you through the steps of quantitative analysis of the steroids data set including importing the existing steroids raw data files into a new workbook folder creating a new Acquisition Sequence Processing Method and Processing Sequence processing the raw data files reviewing calibration standards and unknowns and creating and reviewing reports Although you do not need to create an Instrument Method or an Acquisition Sequence to follow the quantitative analysis procedure for steroids procedures on how to do this are also included in this manual We recommended that you follow the steroids quantitative analysis in the following order 1 Creating a New Study and Workbook on page 22 2 Creating an Acquisition Sequence for the Steroids Example on page 30 3 Editing the Acquisition Sequence for the Steroids Example on page 34 4 Generating a Peak List on page 54 5 Creating a Processing Method on page 71 6 Creating a Processing Sequence on page
73. der You can have as many different workbooks in the same study folder as you want but each workbook must have a unique name This is convenient for organizing your quantitative analysis projects For example you can use one study folder to organize all of the separate quantitative analysis projects that you are doing for a particular client You can not rename the subfolders that LCquan creates in the workbook folder LCquan manages all of the files associated with the project You do not need to remember what individual files are named or where they reside on your hard drive All necessary files are contained within the workbook In addition LCquan uses a file tracking feature to maintain a secure file system Check with your Administrator if you have problems with the security configuration IMPORTANT To maintain secure file operations LCquan tracks the files in the workbook to ensure that none has been modified by an external process If you modify a file in the workbook by using another software application including Xcalibur LCquan will not allow you to reopen the workbook Therefore always use LCquan to open and modify the workbook and its associated files Thermo Scientific 1 Introduction Introduction to Quantitative Analysis with LCquan Acquiring and Processing Data with LCquan Thermo Scientific With LCquan quantitative analysis usually involves the following processes The order of some of these steps is not rigid For e
74. e 6 Calibration levels and associated raw files Calibration Level Raw File cal01 steroids05 cal02 steroids04 steroids15 cal03 steroids03 steroids14 cal04 steroids02 steroids 13 cal05 steroids16 Tip If necessary you can use the sample type and concentration information in the File Information pane at the bottom of the view to identify the data files Tip To display the amount of standard sample associated with a calibration level right click on the calibration level in the Selected Files pane and then choose Show Level Amounts LCquan User Guide 91 5 Creating a Processing Sequence Associating Raw Files with Sample Types Figure 44 Create Sequence Select Files page of the Quantitate view showing the raw files associated with the calibration levels Si L quan Steroids Workbook Eile Zoom View Apps Options Help Tagme SMR COOSCDSO Kt a Create Sequence select files review and edit L_Aeasistion_ Available Files Standards acs Blanks Unknowns D sdk a Selected Files pi m Install Logs TT i methods eo B A QuanRoot oe al Example Study pees H Drugs Workbook oe B Steroids Workbook i aan PEN B one B cal gt a z steroids02 steroids13 samplea Add gt gt _ 3 A ie sampleb samplec lt lt Remove u sampled a samplee Cte steroidsO2 a steroids03 S Gd steroids04 Edt A steroids05 A steroids13 Gi steroids14
75. e Analysis 0 00 7 Using Internal Standards for Quantitative Analysis oaen auauna 8 Sample Types sarraa aei ole bates ch ado bo aa E 9 Standards oat cheated a E eed EE E EE a 9 Unknowns 4h cen er Goths ete Aa e E N weg 9 OGS dake iE es Morale hee aa aad wal eee ale Hee ewe Hee 10 Blanks ance khan soe e Abbe ces Mpiets coin e kis aes coax 10 Introduction to Quantitative Analysis with LCquan 0 10 Overview of LCquatiiin cia chee ay hie es Rope rreren 10 LCquan Folder Sructite ss sac0a ssi deve nwedows ane sawn naw ahaa 16 Acquiring and Processing Data with LCquan 0000 00 17 Usine This Manvel 26 cert eh eke ehh Rhee nee ieee ides 18 LGguan for Ci vib ea ON Le ek pee Be EE Hew ee a Ls 19 Chapter2 Preparing to Perform Quantitative Analysis of Steroids Data 21 Creating a New Study and Workbooks cccscca cca x dae oededas veda sesd 22 Reviewing the Instrument Setup View 556uvini suse saaeseesaseaaes 25 Creating Instrument Methods rie 56k dk be GEA Sek See ee eee 26 Importing an Existing Instrument Method 000 00000 27 Thermo Scientific LCquan User Guide iii Contents Reviewing the Acquisition View ievs 3acatee avant dae baie des 27 Setup Sequence Pages cuss ernea aia a N shane a E 28 Naming Components and Assigning Calibration Levels 36 Run Sequence Dialog Boxee ari 3 a os cio seealel Pace Paen a alesal hares 43 Acquisition View
76. e Filter box the scan filters associated with the raw file In this example full scan MS MS data were acquired on the steroids by using alternating product ion scans To relate the chromatogram peaks with the steroids 1 Select the scan filter for methyltestosterone parent ion mass m z 303 Filter c Full ms2 303 30 40 100 00 310 00 and click Apply LCquan displays the filtered chromatogram See Figure 27 Note that methyltestosterone corresponds to the peak named Peak 1 991 in the peak list Figure 27 Explore view Create Explore Methods page showing a filtered chromatogram for methyltestosterone 6 L quan Steroids Workbook 4 BE q eS File Zoom View Apps Options Help y IF Gaba TEENE E ACA Instruments Identification ears aa Chromatagram Definition Genesis Peak Integration Limit peaks Acquisition Sequence History uantitate Detector Smoothing Peak Detection Algorithm Select top peaks MS iy Genesis Advanced V Enable Trace Select by area S N Threshold 3 TiC Se fe resnowt 10 5 Select by height Mass m 2 C Valley Detection Enabled J4 Expected Width sec 0 0 Num to select GLEE C Constrain Peak Width Rel peak height threshold c Full ms2 303
77. e Review and Explore Results Page Exporting the Active Raw File to Qual Browser LCquan provides a direct link to Qual Browser from the Review and Explore Results page Qual Browser provides for more sophisticated qualitative review than what is available in LCquan which is mainly concerned with quantitative rather than qualitative analysis With Qual Browser you can perform library searches formula searches for spectra background subtraction and other data manipulation To open Qual Browser Click on the Qual Browser button in the viewbar When you open Qual Browser LCquan passes the currently selected raw file to it 70 LCquan User Guide Thermo Scientific Creating a Processing Method Thermo Scientific This chapter describes how to create a Processing Method by using the Create Method pages of the Quantitate view A Processing Method provides instructions to LCquan on how to perform quantitative analysis on raw data A Processing Method contains component identification detection integration and calibration information Contents e Opening the Create Method Page and Using the New Method Wizard e Specifying the Component Identification and Integration Parameters e Jon Ratio Confirmation of Analytes e Importing the Components and Levels into the Acquisition Sequence e Saving the Processing Method e Importing an Existing Processing Method LCquan User Guide n 4 Creating a Processing Method Opening the Create M
78. e locks to compare data from different raw files simultaneously For example to see how the acquisition progressed from sample to sample You can compare a plot from one sample to the plots of other samples by locking one plot and then clicking down the sequence in the sample selector You change the state of the lock by clicking on it The lock can be in three different states e Unlocked green amp Locked against raw file change yellow Absolute lock red When a plot is unlocked every change of raw file or parameter is reflected in the plot Parameters such as retention time are sent out from unlocked plots for example when you click on a chromatogram Plots that are locked against raw file change will respond to changes in parameters but not to a change of raw file Parameters are not sent out from plots that are locked against raw file change Plots that are absolutely locked will not respond to changes in parameters or raw files Parameters are not sent out from plots that are absolutely locked LCquan User Guide 65 3 Exploring Your Data Additional Features of the Review and Explore Results Page General Parameters Plot A general parameters plot provides a graphical representation of the retention time peak area peak height or peak width across the chromatogram A peak area general parameters plot for steroids04 is shown in Figure 32 You can lock the general parameters plot for one sample and click through t
79. e or dialog box Note You have already imported existing raw data into your workbook You do not need to acquire your own data to follow the quantitative analysis procedure described in this manual Thermo Scientific LCquan User Guide 27 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View 28 Setup Sequence Page LCquan User Guide The Acquisition view Setup Sequence page allows you to create or modify an Acquisition Sequence It includes clockwise from the top left the Sequence Header the Acquisition Sequence History pane and the Sequence table See Figure 13 The Acquisition Sequence is a list of samples and defined settings that LCquan uses to acquire data The Acquisition Sequence can contain unknown samples calibration standard samples quality control QC samples and blank samples Each sample can be defined by some or all of the following settings Sample type File name Sample identification QC or Standard level Internal standard correction amount Dilution factor Vial position Injection volume Sample volume Sample weight Sample name Thermo Scientific 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View Figure 13 Acquisition view Setup Sequence page showing an Acquisition Sequence iii FileName Sample Name 2 Standard 3 Standard steroids001
80. een saved in the workbook it remains part of the workbook even if the configured instrument is removed Importing an Existing Instrument Method LCquan also lets you import an existing Instrument Method into your current workbook instead of creating a new method You can use the method as is or edit it if necessary You can import an Instrument Method in the following ways e New Study Wizard When you create a new workbook the New Study Wizard gives you the option of importing Instrument Methods Acquisition Sequences and processing parameters to initialize the new workbook You can select to import all of this data from an existing workbook or you can select to import an Instrument Method from an individual legacy file meth file Instrument Setup view menu command e Instrument Method File dialog box Choose File gt Import Instrument Method to display the Import Instrument Method File dialog box This dialog box allows you to import an Instrument Method from an individual legacy file meth file Reviewing the Acquisition View The Acquisition view allows you to define an Acquisition Sequence run individual samples and sequences and monitor real time data acquisition This view is made up of the following components e Setup Sequence Page e Run Sequence Dialog Box e Naming Components and Assigning Calibration Levels e Acquisition View Status Page You use the viewbar on the left side of the view to select the pag
81. esents the contribution of the internal standard to the chromatogram Tip In the Chromatogram pane and in the ISTD Chromatogram pane you can use the toolbar buttons to zoom in and out along the X and Y axes to show more detail See the online Help for a description of each toolbar button 0000000 Alternatively you can use the arrow keys on the keyboard to zoom The left arrow key zooms in along the X axis by a factor of two 2 The right arrow key zooms out along the X axis by a factor of two after you have zoomed in Similarly the up arrow zooms in along the Y axis by a factor of two and the down arrow zooms out If you want to exclude a calibration standard from the calibration curve go to the next topic Excluding a Calibration Standard from the Calibration Curve Ifyou want to modify the peak detection and integration settings that you defined in the Create Method Identify Components page go to the topic Modifying the Peak Detection and Integration Settings on page 109 If you want to integrate a peak manually go to the topic Integrating Peaks Manually on page 110 You can manually change the integration starting and ending points and the baseline of the peak Inspect the remaining files in the Result List Select the next data file by clicking on the next row in the Result List b Repeat step 8 for each file Thermo Scientific 6 Processing the Raw Files and Reviewing the Analytical R
82. esponse factor 5 Response High note 114 Result List colors in table 102 description 100 Review All Results page illustrated 113 opening 112 using 112 Review and Explore Results page 59 review displays Explore view 69 review mode 22 Review Reports page 116 illustrated 118 LCquan User Guide 131 Index S opening 116 reviewing all results 112 reviewing calibration standards and QCs 102 Run Manager Status 47 Run Sequence dialog box illustrated 43 opening 43 S sample selector Explore view 63 64 sample types blanks 10 quality controls QCs 10 standards 9 unknowns 9 saving workbook 121 scan filters matching with components 77 Select Rawfile dialog box 73 semi quantitative analysis definition 2 sequence See Acquisition Sequence Processing Sequence Setup Sequence page illustrated 29 33 overview 28 Shutdown Methods creating 26 defined 25 Spectrum pane overview 48 Status page 49 Standard Level Names Association dialog box 96 standards described 5 external standards 7 internal standards 8 sample types 9 starting LCquan 22 Startup dialog box 22 Startup Methods creating 26 defined 25 Status page illustrated 46 opening 45 overview 45 status plots Explore view 67 status reports Explore view 68 steroids example 01 creating the study and workbook 22 02 importing raw files 24 03 creating the Acquisition Sequence 30 132 LCquan User Guide 04 editing the Aquisition Sequence 34 05 exploring
83. esults Reviewing the Calibration Curve and OCs 10 Repeat steps 8 and 9 for the remaining target compounds deoxycorticosterone and progesterone The calibration curves for hydrocortisone deoxycorticosterone and progesterone are shown in Figure 50 11 Review and rework the internal standard results as you did for the target compound a Inthe Component List select the internal standard methyltestosterone LCquan automatically updates the Result List and the Chromatogram pane LCquan does not display a chromatogram in the ISTD Chromatogram pane for this component but displays the response for each internal standard sample in the Calibration Curve pane b Check the entries in the Result List for peak detection and integration problems c Inspect the chromatogram in the Chromatogram pane for each file i Ensure that LCquan found the peak ii Ensure that the shaded area accurately represents the contribution of the component to the chromatogram e Ifyou want to modify the peak detection and integration settings that you defined in the Create Method Identify Components page go to the topic Modifying the Peak Detection and Integration Settings on page 109 e Ifyou want to integrate a peak manually go to the topic Integrating Peaks Manually on page 110 You can manually change the integration starting and ending points and the baseline of the peak When you are satisfied that LCquan detected and integrated the peaks
84. ethod Page and Using the New Method Wizard Opening the Create Method Page and Using the New Method Wizard In this example you need to open the Create Method page of the Quantitate view to construct a Processing Method When you open this page the New Method Wizard appears and leads you through the initial steps of creating a method To open the Create Method page of the Quantitate view and start the New Method Wizard 1 From any view click on the Quantitate button in the LCquan viewbar The New Method Wizard appears See Figure 35 2 Click on Next on the Welcome page of the wizard to continue 3 Select the Create New Method option button the Initialize with Acquisition Component Names check box and click on Next Note The Initialize With Acquisition Component Names check box is unavailable if you have not yet defined component names Figure 35 New Method Wizard Welcome page New Method Wizard Welcome to the New Method Wizard LCqua n 7 5 This wizard helps you setup a new processing method A New Era in Mass Spectrometry 72 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Opening the Create Method Page and Using the New Method Wizard 4 Select the Calibrate Using Internal Standards option button to specify that you are calibrating by using an internal standard Then click on Next 5 Open a raw file from your data set to test the peak detection and integration parameters Note In ge
85. ethod into the Acquisition Sequence Although it is not necessary to do this for the steroids example the procedure is described below To import the component names and calibration levels into the Acquisition Sequence Acquisition Sequence 1 Click on the Acquisition button and then the Setup button to display the 2 Right click on the Sequence grid and select Standard and QC Levels from the shortcut menu to display the Acquisition Levels dialog box 3 Click on Import Processing Components and then on OK to import the component names and calibration levels from the Processing Method See Figure 41 Figure 41 Acquisition Levels dialog box showing the steroids components and calibration levels Acquisition Levels Components Levels component Cattever amount methyltestosterone cctevet amount wrest N Import Processing Components Load Default Levels OK 84 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Saving the Processing Method Saving the Processing Method When you have finished creating the Processing Method choose File gt Save to save the settings in the workbook For information on other ways to define a Processing Method in a new workbook go to the next topic Importing an Existing Processing Method Otherwise go to Chapter 5 Creating a Processing Sequence Importing an Existing Processing Method When you are doing routine quantitative analysis e
86. f Quantitative Analysis by LC MS MS e Quantitative Analysis Techniques e Sample Types e Introduction to Quantitative Analysis with LCquan e LCquan for Citrix lFor further information on the principles of quantitative analysis refer to the following Mass Spectrometry Principles and Applications de Hoffman E Charette J Stroobant V Wiley New York 1996 and Introduction to Mass Spectrometry 3rd ed Watson J T Lippincott Raven Philadelphia PA 1997 Thermo Scientific LCquan User Guide 1 1 Introduction About Quantitative Analysis About Quantitative Analysis Quantitative analysis is the process of measuring the amount of a particular component in a sample In some applications such as a clinical trial you might be seeking the maximum possible accuracy from your measurements Time and cost of analysis are less important than achieving the highest possible standards in precision and accuracy This form of measurement is called quantitative analysis In other applications such as in trace analysis you might only want to estimate the quantity of a component It might be sufficient to know that the component is present at a level either significantly higher or significantly lower than a defined threshold For example it is generally not important to know whether a patient has overdosed 15 or 20 times above a prescribed limit but simply that the limit has been exceeded In such cases a rapid measurement is required
87. faced to an LC should consider the following variables to optimize detection of ions by MS MS e Solution chemistry and polarity of the analyte of interest e Probe selection H ESI ESI APPI or APCI e Collision energy to fragment parent ions inside the mass spectrometer optimized by experiment The LC provides a stream of analyte solution eluate into the mass spectrometer where the analyte is detected Consideration must be given to the content of modifiers salts and contaminants in the eluate Specifically you must ensure that what goes into the inlet of the mass spectrometer does not suppress ionization of the compound of interest Salt concentrations above 10 mM and strong acids and bases will damage the LC column Modifier concentrations greater than 10 mM are not usually necessary for chromatographic stability and can suppress ionization of other compounds Whenever possible it is best to use volatile modifiers Volatile modifiers include the following e Acetic acid e Ammonium acetate e Ammonium formate e Ammonium hydroxide e Formic acid Trifluoroacetic acid Your analyte solution can contain neutral particles or ions If the solution contains ions the ions can carry a single charge or multiple charges The number of charges depends on the structure of the analyte and the composition and pH of the mobile phase Solvent systems are generally composed of organic solvents water and volatile modifiers The ionization char
88. fferent views of LCquan The following flow diagrams are contained in this appendix e LCquan flow diagram e LCquan for Citrix flow diagram e Acquisition view flow diagram e Quantitate view flow diagram Thermo Scientific LCquan User Guide 123 A LCquan Flow Diagrams Figure 57 LCquan flow diagram showing the steps for creating a new study or workbook and the four main views in dark gray LCquan Flow Diagram Create New Study or Create New Study or Workbook Workbook or Open Existing Workbook Study and Workbook Names Page Click on Next Import Raw Files Import from Legacy Files Instrument Method Click on Next Files Page Acquisition Sequence Import from and Existing Workbook Select Workbook Click on Next Processing Parameters Click on Next Page and Finish Page Click on Next and Finish DoNot Clickon iis ye Import Next amp Method Prior Data Finish Imported Basic Instrument Method Created Instrument Method Updated for Configured for Configured Instruments Instruments Instrument Setup View Acquisition View See Acquisition Flow Diagram Click on Method button j Create Explore Method Page Click on Review button Click on Setup button Setup Sequence Page Click on Acquire button y Run Sequence Dialog Box Click on OK Review the Explore Results Page 124 LCquan User Guide Open Existing
89. has been designed for UV data Avalon also supports negative peaks You have finished entering the component identification and integration settings for the target compounds and the internal standard Review the integrated peaks again e Ifthe peaks have been integrated correctly go to the next topic Ion Ratio Confirmation of Analytes e Ifthere is excessive noise in a peak unresolved peaks or peak tailing return to the topic Entering the Component Identification Parameters and Integrating the Peaks on page 77 and change the peak identification and integration parameters e Ifbaseline noise is interfering with peak identification or integration change the settings in the Advanced Parameters dialog box See the online Help for further information 82 LCquan User Guide Thermo Scientific lon R 4 Creating a Processing Method lon Ratio Confirmation of Analytes atio Confirmation of Analytes Ion ratio confirmation IRC allows you to confirm the presence of an analyte by comparing the response of the quantitate ion or ions with the responses of one to five qualifier ions LCquan marks the analyte as Not Found in the Results List of the Quantitate view if any of the qualifier ion quantitate ion response ratios are outside of the upper or lower bounds that you specify or if the quantitate and qualifier ions do not coelute You specify the masses target area ratios and area ratio windows of the qualifier ions in the Io
90. he other samples to look for trends or inconsistencies in the data Figure 32 General parameter plot top and corresponding chromatogram bottom for steroids04 dra General Parameters Pli steroids04 3800000 3600000 3400000 3200000 3000000 A o 2800000 unt 82600000 a 2400000 2200000 2000000 1800000 1600000 0 675 1 383 steroids04 TIC SM 7 RT 0 01 3 79 NL 9 59E5 100 RT 0 68 90 80 70 60 50 40 gt pa o o 2 E o 30 1 991 3 178 Peak Chromatogram 249 2 73 2 0 Time min 66 LCquan User Guide Thermo Scientific Status Plot Thermo Scientific 3 Exploring Your Data Additional Features of the Review and Explore Results Page A status plot displays status data that is stored by instruments during the acquisition of the selected raw file The drop down list lists all the parameters for the selected instrument You can use the arrow buttons to move up or down the list A status plot of the capillary temperature is shown in Figure 33 Status plots can be used to investigate an anomaly in a chromatogram or to verify that the Instrument Method did what you expected it to do Figure 33 Status plot of the capillary temperature Capillary Temp C Status Plot steroids04 Device MS 175 6 1754 a ho a o m oO a E o BE gt ra S a 5 oO 2
91. ide of the page is one pane defaulted to Multi Peak Plot Comparison View Displays three stacked panes defaulted to Chromatogram Spectrum and Peak List Peak List View Displays side by side two tall panes defaulted to Chromatogram and Peak List Sample Info View Displays four panes one pane in the top row and three small side by side panes in the bottom row defaulted to Chromatogram Sample Info Instrument Method List and Status Plot Large Chro View Displays a large single pane defaulted to Chromatogram Note You can change the contents of the panes but not their sizes shapes and positions To change the name of the layout Select the name in the Review Display list and then type in the new name To import a layout Choose Files gt Layout Configuration gt Import Layout Configuration to display the LCquan Layout Config dialog box Then browse to the LCquan config file and click on Open LCquan places the config file in the Imports folder To save a layout Choose Files gt Layout Configuration gt Export Layout Configuration to display the LCquan Config File dialog box Then enter a name for the config file and click OK LCquan places the config file in the Exports folder Note If you change the properties of a display and save the workbook the new properties will be active when you reopen the workbook Thermo Scientific LCquan User Guide 69 3 Exploring Your Data Additional Features of th
92. iew 17 procedure 44 saving workbook after note 45 Acquisition Levels dialog box illustrated 36 37 41 using 41 Acquisition Queue 47 Acquisition Sequence copying Processing Sequence to 38 copying to Processing Sequence 96 creating 30 definition 28 editing 39 exporting 42 illustrated 35 importing 38 printing 42 Acquisition Sequence History 48 63 Acquisition view opening 30 overview 27 Acquisition view Setup Sequence page See Setup Sequence page Acquisition view Status page See Status page analyte detection using mass spectrometry 4 associating raw sample files with sample types 89 audit trail Explore view 52 Avalon peak detection 77 blanks 10 C calibration curve curve types 15 described 5 Thermo Scientific excluding standards 107 inspecting at low concentrations 102 steroids figure 106 using external standard 7 using internal standard illustrated 9 weighting 15 Calibration Curve pane 102 calibration curve selecting points 103 calibration curve showing only low concentration points 103 calibration levels assigning 36 41 associated steroids raw files and table 91 matching names note 32 32 38 calibration levels and amounts for steroids 34 calibration standards 9 Chromatogram pane overview Acquisition Status view 48 in Survey page 102 using Review All Results page 114 Status page 48 Chromatogram pane and Spectrum pane Acquisition view 50 Citrix LCquan for 19 colors in Result List 102
93. illustrated 61 exporting component names 56 generating 54 illustrated 55 renaming peaks 61 peaks integrated illustrated 6 integrating manually 110 modifying integration parameters 109 properties viewing 114 undoing manual integration of 110 printing Acquisition Sequence 42 probes API selecting 4 processing overview 17 raw sample files 99 110 while acquiring data 50 Processing Method creating 71 defined 71 importing 85 saving 85 Processing Sequence copying Acquisition Sequence to 96 copying to Acquisition Sequence 38 correcting errors in note 94 creating 87 defined 87 editing 97 Thermo Scientific Index N importing 95 reviewing 94 progesterone calibration curve figure 106 Q QC Level Names Association dialog box 96 QC levels assigning 36 41 matching names note 32 32 38 Qual Browser starting from Explore view 70 quality control QC samples 10 quan components creating from peak list 56 61 Quantitate Sequence 63 quantitation limits 6 quantitation range 6 quantitative analysis described 2 10 LCquan overview 17 sources of error 8 steps of 2 techniques 5 using external standards 7 using internal standards 8 raw sample files associating with sample types 89 displaying in Chromatogram pane 48 importing into new workbook 24 locked and unlocked 65 processing 99 110 Realtime Display Settings dialog box 49 reports creating 116 118 Microsoft Excel 116 options 119 XReport templates 116 r
94. ing or ending point of a peak position the cursor over one of the integration markers The cursor turns into crosshairs Then click and drag the integration marker to the desired location e By using the Baseline dialog box To precisely position a starting or ending point of a peak right click in the Chromatogram pane to display the shortcut menu Choose Change Peak Baseline to open the Baseline dialog box Enter the new coordinates for the starting and ending points into the appropriate text boxes and click on OK When you integrate a peak manually LCquan displays Manual Integration in the Integration Type column of the Result List See Figure 52 You can easily undo the manual integration by using the Integration Type column of the Result List In the Integration Type column click in the cell containing the words Manual Integration for the sample that you want to change Then click on the down arrow and select Method Settings from the drop down list to return the integration to its prior settings 110 LCquan User Guide Thermo Scientific 6 Processing the Raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Figure 52 Survey page of the Quantitate view showing a manually integrated peak in the Chromatogram pane A L quan Steroids Workbook File Zoom Yiew Apps Options Help Manual Integration Type saan HAODD Ki ey review standards and QCs riene
95. ing the Save Only or the Print and Save option LCquan places a copy of the report in the Exports folder of the workbook LCquan User Guide 117 6 Processing the Raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Figure 56 Review Reports page of the Quantitate view showing the report options 40 LCquan Steroids Workbook fe fel File Zoom View Apps Options Help ss USHAL COGOGSOSOSO Ki OO Cn Feel Hever Serine Manage Excel Column Arrangements Column Arrangement Select an Arrangement E xcel Long Summary x Sample Style Excel Report a oi Eves Renate Launch XReport Template Generator Create XReport Templates Report Report Selections Select Report Options Print Only O Save Only Print and Save For Help press F1 NOT SAVED 8 2 2005 3 21 P 118 LCquan User Guide Thermo Scientific 6 Processing the Raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Table 8 Report options available in Review Reports page Sheet 1 of 2 Report Options x XReport Selections Description Acquisition Sequence Report LCquanAcqSequence xrt Displays the sample information that is in the Acquisition Sequence for example sample ID raw file names sample types and cal levels Component Calibration Report Avalon Genesis or ICIS algorithm LCquanCompCalReport xrt Provides the component identification and calibration p
96. ion Curve Parameters for the Target Compounds 75 Entering the Component Identification Parameters and Integrating the Peaks77 Ion Ratio Confirmation of Analytes 3 20 sh ah09s3an 2h 96e 08 erae 83 Importing the Components and Levels into the Acquisition Sequence 84 Saving the Processing Method 3 ic 45 04 visas ees worse nne 85 Importing an Existing Processing Method 0000 c eee eee 85 Chapter5 Creating a Processing Sequence 0eeee eee eee eee eens 87 Opening the Create Sequence Page and Using the New Processing Sequence Wiad gre yeaa dish ats Ate Grae wn eae ee eee E a 88 Associating Raw Files with Sample Types 0 0 eee eee eee 89 Reviewing the Processing Sequence n aa wesw eheo s sade wan ws 94 Further Information Additional Ways to Define the Processing Sequence 95 Importing an Existing Sequence 0 cece eee 95 Copying the Acquisition Sequence 20 cee eee cee eee 96 Further Information Editing the Processing Sequence 0000 97 iv LCquan User Guide Thermo Scientific Thermo Scientific Contents Chapter6 Processing the Raw Files and Reviewing the Analytical Results 99 Processing the Raw Files 5 2 acsabi vate ld a neee 100 Reviewing the Calibration Curve and QCs i 6 deed eed dees a ees 100 Opening the Survey Page nds ious cas Bond wake alas Ae ee ae eed 100 Excluding a Calibration Standard from the Calibration Curve 107
97. is Acquisition Sequence associated raw files with calibration levels or unknowns and specified that there were four components and five calibration levels You use the Create Sequence Select Files page to build a Processing Sequence by associating each raw file in the data set with one of the following sample types standard QC blank or unknown You then associate the standards and QCs with the appropriate calibration level or quality control level To build anew Processing Sequence for the steroids example 1 Locate the steroids raw data set a In the directory tree in the Available Files pane browse to find the Rawfiles folder in your current workbook In this example the default path is Xcalibur QuanRoot Practice Study Steroids Workbook Rawfiles b Click on the Rawftles folder LCquan displays in the Available Files pane all of the steroids raw files See Figure 43 Thermo Scientific LCquan User Guide 89 5 Creating a Processing Sequence Associating Raw Files with Sample Types Figure 43 Create Sequence Select Files page of the Quantitate view showing the raw data files in the steroids data set Si LC quan Steroids Workbook EEk File Zoom View Apps Options Help DeHS AL 29h C00OOOO xu i rens Create Sequence select files review and edit Available Files xplore Standards QCs Blanks Unknowns Ge Heb 2 Selected Files
98. is techniques e External standard quantitative analysis e Internal standard ISTD quantitative analysis The chosen method determines the manner in which response is calculated both for the generation of the calibration curves and for subsequent quantitative analysis These methods are described in the following topics e Using External Standards for Quantitative Analysis e Using Internal Standards for Quantitative Analysis Thermo Scientific 1 Introduction Quantitative Analysis Techniques Using External Standards for Quantitative Analysis Thermo Scientific An external standard is a separate sample that contains the compound of interest at a known concentration in solution In the external standard quantitative analysis technique a series of standards are analyzed and a calibration curve is constructed by plotting the magnitude of the detector response as a function of the external standard concentration The unknown sample is then analyzed and the concentration is determined by matching the magnitude of the detector response with that on the calibration curve See Figure 2 Using external standards is advantageous if various components in a sample are being analyzed and if all compounds of interest can be assayed by using a single set of external standards This approach offers time and cost effective quantitative analysis although it is not capable of achieving the very highest precision and accuracy Variations in analyte and
99. isting steroids data This chapter also describes the Instrument Setup view and the Acquisition view of LCquan Although the step by ste P q q 8 y procedure for performing quantitative analysis contained in this manual does not require that you create Instrument Methods or acquire your own data this chapter outlines the procedures for doing this Contents e Creating a New Study and Workbook e Reviewing the Instrument Setup View e Reviewing the Acquisition View Thermo Scientific LCquan User Guide 21 2 Preparing to Perform Quantitative Analysis of Steroids Data Creating a New Study and Workbook Creating a New Study and Workbook When you start LCquan you can open an existing study and workbook or you can create a new study and workbook set You can also choose to open a workbook in review mode In review mode you can analyze existing data and create methods but you cannot acquire data For the quantitative analysis of steroids data you create a new study and workbook and import raw steroids data to start your analysis To create a new study and workbook 1 Open LCquan from your desktop by choosing Start gt Programs gt Xcalibur gt LCquan LCquan displays a startup dialog box from which you can open the last used workbook open an existing workbook or create a new workbook See Figure 9 If you have used LCquan previously the name of the most recently opened workbook appears directly above the buttons In addition the Mo
100. ive Intensity a For Help press F1 76 robert heather 2 Specify the calibration curve parameters for deoxycorticosterone a Select deoxycorticosterone in the Component list b On the Calibration page select or enter the following LCquan User Guide e Component Type Target Compound e Target Units ng e Calibration Curve Type Linear e Response Area e Origin Include e Weighting Equal NOT SAYED 11 7 2005 2 36 PI Thermo Scientific 4 Creating a Processing Method Specifying the Component Identification and Integration Parameters c Click on Apply to apply the settings 3 Specify the calibration curve parameters for progesterone a Select progesterone in the Component list b On the Calibration page select or enter the following e Component Type Target Compound e Target Units ng e Calibration Curve Type Linear e Response Area e Origin Include e Weighting Equal c Click on Apply to apply the settings Go on to the next topic Entering the Component Identification Parameters and Integrating the Peaks Entering the Component Identification Parameters and Integrating the Peaks When LCquan acquires data it creates unique scan filters according to the type of experiment you specify in the Instrument Method When you load a raw file LCquan lists in the Filter combo box the scan filters associated with the raw file In this example full scan MS MS data were acquired
101. kbook A New Era in Mass Spectrometry oreo 5 You can initialize a workbook by importing data from another workbook or from legacy files Note LCquan does not permit you to overwrite existing data If you attempt to name a new study and workbook set the same as an existing set LCquan displays the following message box Click OK and then type another study or workbook name Thermo Scientific LCquan User Guide 23 2 Preparing to Perform Quantitative Analysis of Steroids Data Creating a New Study and Workbook 6 Select the raw data files to import into the new workbook a Select the Import Rawfiles check box b Click on Browse to open the Select Rawfile dialog box c Browse to Xcalibur examples data d Click on samplea and then hold down SHIFT and click on steroids17 to select the 14 steroid raw files See Figure 11 e Click on Open and then Next to continue Figure 11 Select Rawfile dialog box showing the selected steroids raw files Select Rawfile Look in B My Recent Documents e Desktop My Documents My Computer My Network data drugx_29 ii drugx_30 iia drugx_31 Full_scan_apiginin_ms2 i Full_scan_Ms ia Full_scan_M52_579 iia Full_scan_ms3_271 Full_scan_ms3_433 Full_scan_ms4_271 lidJinfusion i msnbrowser i nimap3 G pmap3 idjreserpineos ijreserpineo4 iiaJreserpineos EESE lidJreserpineos F steroids17 reserpine ligltimap3
102. le Study C Xcalibur QuanRoot Example Study LCquan Internal Sample04 steroids003 C Xcalibur QuanRoot Example Study C Xcalibur QuanRoot Example Study LCquan Internal Sample0S steroids004 C XcaliburiQuanRoot Example Study C XcaliburiQuanRoot Example Study LCquan Internal Sample13 steroids005 C Xcalibur QuanRootExample Study C XcaliburiQuanRoot Example Study LCquan Internal Sample14 steroids006 C Xcalibur QuanRoot Example Study C Xcalibur QuanRoot Example Study LCquan Internal Sample15 steroids007 C Xcalibur QuanRootExample Study C Xcalibur QuanRoot Example Study LCquan Internal Sample16 steroids008 C Xcalibur QuanRoot Example Study C XcaliburiQuanRoot Example Study LCquan Internal Samplea steroids009 C Xcalibur QuanRoot Example Studyt C Xcalibur QuanRoot Example Study LCquan Internal Sampleb steroids010 C XcaliburQuanRootiExample Study C XcaliburiQuanRoot Example Study LCquan Internal Samplec steroids011 C Xcalibur QuanRoot Example Study C Xcalibur QuanRootExample Study LCguan Internal Sampled steroids012 C Xcalibur QuanRootExample Study C Xcalibur QuanRoot Example Study LCquan Internal Samplee steroids013 C Xcalibur QuanRoot Example Study C Xcalibur QuanRoot Example Study LCquan Internal
103. lgorithm Genesis e Filter c Full ms2 303 30 40 100 00 310 00 scan filter for m z 303 parent mass c Click on Apply to apply the settings LCquan applies the settings to the data in the raw file and automatically displays the resulting filtered chromatogram in the Chromatogram pane Specify the expected retention time of methyltestosterone a Inspect the mass chromatogram and note the retention time of methyltestosterone Each component peak is labeled with the appropriate retention time In this example the methyltestosterone peak is labeled R7 1 98 b In the Expected text box type 1 98 to specify that the retention time of methyltestosterone is 1 98 min Tip Two commands in the Chromatogram pane shortcut menu can help you specify the retention time for a component peak Set Spectrum To Peak Apex and Auto Update Expected RT From Spectrum Marker Right click anywhere in the Chromatogram pane to display the shortcut menu Choose Auto Update Expected RT From Spectrum Marker to turn on the automatic updating feature for the expected retention time When this feature is on LCquan automatically enters the time indicated by the spectrum marker into the Expected text box in the Retention Time group box Choosing this command again turns off this feature Choose Set Spectrum To Peak Apex to automatically place the red spectrum marker at the peak apex and enter the retention time in the Expected box Thermo Scientific Ther
104. mo Scientific 4 Creating a Processing Method Specifying the Component Identification and Integration Parameters c In the Window text box type 30 00 to specify that the peak detection time search window is 30 00 s wide centered about the retention time d Select the Use As RT Reference check box to use the retention time of methyltestosterone to adjust for the retention time drift of other components e In the View Width text box type 0 75 to specify a chromatogram view width of 0 75 min f Click on Apply to save the component identification information for methyltestosterone 4 Integrate the methyltestosterone chromatogram peak a Click on the chromatogram peak in the Chromatogram pane b Click on Apply to integrate the chromatogram peak LCquan automatically integrates the peak according to the specified parameters The integrated portion of the peak is shaded gray and blue identification markers are displayed at the starting and ending points of the peak integration The blue line that connects the integration markers indicates the baseline See Figure 39 Inspect the integrated peak in the Chromatogram pane and verify that the following are true e The peak has the proper symmetry and the grayed area accurately represents the contribution of the component to the chromatogram e The entire chromatogram peak is displayed in the Chromatogram pane If necessary increase the view width by entering a larger value in the
105. mple Study C Xcalibur QuanRoo steroids015 cal05 C XcaliburQuanRootiExample Study C Xcalipur QuanRoc v For Help press F1 m E NOT SAYED 10 26 2005 12 52 PM Thermo Scientific Each row of the sequence corresponds to one sample injection You need an Acquisition Sequence to acquire data and a Processing Sequence to process data The Acquisition Sequence and the Processing Sequence can be the same Each sequence has a limit of 5000 samples The following subtopics describe how to use the Setup Sequence page e Creating an Acquisition Sequence for the Steroids Example e Editing the Acquisition Sequence for the Steroids Example e Importing or Copying an Existing Sequence e Editing an Acquisition Sequence e Assigning Calibration or QC Levels e Printing and Exporting the Acquisition Sequence LCquan User Guide 29 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View Creating an Acquisition Sequence for the Steroids Example Although you do not need to create an Acquisition Sequence to follow the quantitative analysis procedure described in this manual the procedure is outlined below For more information refer to the online Help 1 To define an Acquisition Sequence for the steroids example 1 From the Instrument Setup view see previous section click on the Acquisition bu
106. n ISTD should be closely related to the target component in terms of its physical and chemical properties It must be pure not present in the sample and inert towards the components of the sample ISTD components are typically analogs homologues or isomers of the target non ISTD component An ideal ISTD is a structural or isotopically labeled analog of one of the target components Stable isotope labeled ISTDs act almost identically to the Thermo Scientific Sample Types Standards Unknowns Thermo Scientific 1 Introduction Sample Types analyte throughout sample manipulation and with regard to ionization tendencies and fragmentation Internal standards labeled with two or more deuterium D atoms are frequently used for LC MS There can be any number of ISTD components in a sample but each non ISTD component can be calibrated against only one ISTD Figure 3 Calibration curve generated by using an internal standard Response Ratio for 1 2 4 Unknown Sample Unknown A M 0 0 r r r i 0 0 0 5 1 0 1 5 2 0 25 Amount of Target Compound A Response for Target Compound Response for ISTD Fach quantitative analysis consists of a number or sequence of samples The sequence represents the order of sample analysis A quantification sequence contains One or more standards One or more unknown samples For more demanding applications you can also use optional quality control QC samples and blank samples
107. n Ratio Confirmation table of the Processing Method LCquan displays the mass chromatograms of the qualifier and quantitate ions in the IRC Chromatogram pane In the example shown in Figure 40 LCquan will mark hydrocortisone as Not Found if the area of the m z 327 2 mass chromatogram peak divided by the area of the m z 309 2 267 2 mass chromatogram peak and multiplied by 100 is less that 50 or greater than 70 Also the peak maximum of the qualifier ion chromatogram must be within 0 1 min of the peak maximum of the quantitate ion chromatogram Figure 40 lon ratio conformation settings for hydrocortisone using m z 327 1 as the qualifier ion A LCquan St File Zoom View Apps GaH Quantitate eroids Workbook Options Help DER LELeVR COOSCCOO KHL OVO Identification Calibration Chromatogram Definition Genesis Peak Integration Peak Identification deoxycotticosterone Detector Smoothing Peak Detection Algorithm metnytesiosterone MS J 7 Gen sis x Highest Peak Min Peak Height S N Progesterone 7 O Nearest RT 3 0 ieee S N Threshold 0 5 reshold v v Mass Range J lon ratio confirmation Mass m z Valley Detection Enabled M Enabled lon ratio using Area 267 2 309 2 EEESSS J Mr fit TER Target Window Fitter od Ratio c Full ms
108. n and Xcalibur and choose Start gt Programs gt Xcalibur gt Instrument Configuration to open the Instrument Configuration dialog box 2 Ensure that the Instrument Method instrument Method button is selected 3 Enter the instrument settings that are appropriate for the experiment that you want to perform Note that the Instrument Setup view might contain multiple pages or dialog boxes 4 Repeat steps 1 through 3 for each instrument that you want to set up 5 If you want to create an optional Startup Method that LCquan will run before it runs the first sample in an acquisition do the following a Select the appropriate instrument by clicking on its icon in the viewbar b Click on the Startup Method button c Enter the startup settings for the instrument 6 If you want to create an optional Shutdown Method that LCquan will run after it runs the last sample in an acquisition do the following a Select the appropriate instrument by clicking on its icon in the viewbar b Click on the Shutdown Method button c Enter the shutdown settings for the instrument If you create or modify an Instrument Method LCquan automatically saves it when you leave the Instrument Setup view The file name is based on the workbook name for example Steroids Workbook meth 26 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View After a method has b
109. neral open a raw file that corresponds to a low concentration calibration standard a Click on Browse b Browse to find the steroids04 raw file in the Rawfiles folder in your new workbook See Figure 36 In this example the default path is Xcalibur QuanRoot Practice Study Steroids Workbook Rawfiles steroids04 raw c Click on Open d Click on Next 6 Click on Finish to exit the New Method Wizard Figure 36 Select Rawfile dialog box showing steroids04 raw selected Select Rawfile Look in G Rawfiles H samplea id steroids03 iia steroids 16 M sampleb iin steroids17 G samplec lia steroidsoS li sampled ii steroids 13 la samplee id steroids14 a steroidsO02 id steroids 15 File name steroids04 Files of type Raw Files raw Header Information The Create Method Identify Components page of the Quantitate view opens See Figure 37 You are now ready to begin building the Processing Method Thermo Scientific LCquan User Guide 73 4 Creating a Processing Method Opening the Create Method Page and Using the New Method Wizard 74 File Zoom Yiew Apps Options Help Figure 37 Create Method Identify Components page of the Quantitate view ds Workbook HDeHaARLELEPR COEOSCOCOOKHLBO
110. ng Sequence You can import an existing sequence into your current workbook in the following ways e By using the New Study Wizard When you create a new workbook the New Study Wizard gives you the option of importing Instrument Methods Acquisition Sequences and processing parameters to initialize the new workbook You can select to import all of this data from an existing workbook or you can select to import an Acquisition Sequence from an individual legacy file s d ipp or csv file or from an LCquan file gz file If you import an Acquisition Sequence from an individual legacy file into a workbook that does not contain a Processing Sequence LCquan uses the same sequence for the Acquisition Sequence and the Processing Sequence in the current workbook e By using the New Processing Sequence Wizard in the Quantitate view When you use the New Processing Sequence Wizard you have the option to import a previously saved sequence You can import a sequence from an individual legacy file sld ipp or csv file or from an LCquan file gz file By using menu commands from within the Quantitate view From the Create Sequence pages of the Quantitate view you can choose File gt Import gt Processing Sequence gt From File to display the Import Sequence File dialog box This dialog box allows you to import a sequence from an individual legacy file sld ipp or csv file or from an LCquan file gz file You can al
111. ns E My Computer 5 elected Files S Control Panel ki J 3 Floppy 4 Sorbie W s Local Disk C samplec 4 CD RW Drive D sampled S GETXcalHLP on Ussjo sanserv1 W samplee S TechPubs Archive on Ussjo sanserv1 X S F TechPubs on Ussjo sanserv1 Y 13 My Network Places E My Documents og Recycle Bin S XCALI 97106_Getting Productive Quantitation with LC Quan Add gt gt Help Edt Field Value The file raw could not be Found or is invalid For Help press F1 __ __ 10 31 2005 4 08 Ph Thermo Scientific LCquan User Guide 93 5 Creating a Processing Sequence Reviewing the Processing Sequence Reviewing the Processing Sequence After you associate all of the raw data files with sample types you need to review the Processing Sequence to ensure that it is correct To review the Processing Sequence 1 Click on the Edit button to open the Create Sequence Review and Edit page and to display the new Processing Sequence 2 Inspect the Processing Sequence and ensure that each raw file in the steroids data set is properly associated with a calibration level or QC level or is defined as an unknown sample See Figure 46 Note To correct errors in the Processing Sequence click on the Create button to return to the Create Sequence Select Files page Then use the Remove and Add buttons to reassociate the raw files When you are finished
112. ntitative Analysis of Steroids Data Reviewing the Acquisition View When you select a component in the Component table the shortcut menu contains the command Copy Current Component Levels to All Target Compounds to copy the level settings for the selected component to all other components or Load Default Levels for Current Component to load default levels for the selected component To access the shortcut menu right click anywhere in the dialog box After you define components and levels in the Acquisition Levels dialog box this information can be transferred into the Quantitate view to use when you create your Processing Method and Processing Sequence Printing and Exporting the Acquisition Sequence You can print the Acquisition Sequence information in several formats You can print a brief listing of the samples arranged in order of the autosampler vial position You can print the entire sequence with all of the columns Or you can print the sequence with only the columns that are displayed in the Sequence table To print choose File gt Print Sequence Info The Print Selection dialog box opens and allows you to select the format of the printing output In addition you can export the Acquisition Sequence To export choose File gt Export Acquisition Sequence The Save As dialog box opens and allows you to save the sequence file sld When you export a file LCquan places a copy of the file in the Exports folder for the workbook 42
113. o 16 Unknown sampleb NA sample007 C XcaliburiQuanRootlExample Study C Xcalibur QuanRoo Unknown samplec NA sample008 C Xcalibur QuanRootExample Study C Xcalibur QuanRoo ja Unknown sampled NA sample009 C XcaliburiQuanRootiExample Study C Xcalibur QuanRoo ja Unknown samplee NA sample010 C XcaliburiQuanRoot Example Study C XcaliburQuanRoo 0 Standard steroids13 cal04 C Xcalibur QuanRootExample Study C Xcalibur QuanRoo Standard steroids14 cal03 C XcaliburiQuanRootlExample Study C Xcalibur QuanRoo 12 Standard steroids15 cal02 C XcaliburiQuanRootlExample Study C Xcalibur QuanRoo 413 Standard steroids16 cal05 C XcaliburiQuanRootlExample Study C Xcalibur QuanRoo Sample identification v m gt NOT SAVED 10 26 2005 2 45 PI For Help press F1 3 Choose File gt Save to save the Acquisition Sequence and workbook To continue with the steroids example continue with the next topic Naming Components and Assigning Calibration Levels Thermo Scientific LCquan User Guide 35 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View Naming Components and Assigning Calibration Levels You can change the component names and define or modify the calibration levels and QC levels that the Acquisition Sequence uses by using the Acquisition Levels dialog box To change the component names and define o
114. on Sequence from an individual legacy file into a workbook that contains a Processing Method but does not contain a Processing Sequence LCquan uses the same sequence for both Note When the imported Acquisition Sequence is also used as the Processing Sequence the level names must match those defined in the Processing Method If the calibration or quality control level names in the sequence are different from those in the Processing Method LCquan displays the Standard Level Names Association dialog box of the QC Level Names Association dialog box You can use these dialog boxes to associate the level names in the sequence with the level names in the Processing Method If the imported sequence contains calibration or QC levels but the Processing Method does not LCquan displays a warning box to inform you It then displays the Sample Type Assignment dialog box You can select to change the sample type of these samples to unknown in the Processing Sequence or to discard the samples that have these levels in the Processing Sequence e By using the New Acquisition Sequence Wizard in the Acquisition view When you use the New Acquisition Sequence Wizard in the Acquisition view you have the option to import a previously saved file You can import a sequence from an individual legacy file s d ipp or csv file or from an LCquan file gz file e By using menu commands from within the Acquisition view From the Setup Sequence page of the Ac
115. on steroids by using alternating product ion scans To quantitate hydrocortisone deoxycorticosterone and progesterone you need to filter the chromatogram You use the Peak Detection Algorithm list box which is found in the Peak Identification group box to specify the type of peak detection algorithm ICIS Genesis or Avalon to be used to analyze the raw data These algorithms apply smoothing construct a chromatogram using the scan or mass filters assign peak numbers generate a peak list and determine the peak start and peak end points All algorithms provide component peak detection and chromatographic peak detection Thermo Scientific LCquan User Guide 77 4 Creating a Processing Method Specifying the Component Identification and Integration Parameters 78 LCquan User Guide To specify the peak detection algorithm scan filter and the masses of the significant product ions that you determined in the Explore view for each of the steroids Click on the Identification tab to display the Identification page Enter the settings for methyltestosterone a Select methyltestosterone from the Component list b Enter or select the following settings e Detector MS e Trace Mass Range specifies a mass range not TIC chromatogram e Masses 267 3 285 1 important masses that you determined in the Explore view e Smoothing 7 Seven points are to be used in the moving average for smoothing the data e Peak Detection A
116. orm Quantitative Analysis of Steroids Data Reviewing the Acquisition View You can change a range of cells by using the Sequence Fill Down dialog box This dialog box allows you to fill selected rows of selected columns with duplicate text entries or appropriately sequenced number entries Right click in the sequence to display a shortcut menu Then choose Fill Down to open the Sequence Fill Down dialog box You can also use common keyboard shortcuts to edit the sequence CTRL C copies the selected row CTRL V pastes a copied row and DELETE removes the selected row from the sequence Finally the shortcut menu which is displayed by right clicking in the Sequence table contains commands that allow you to delete rows to insert rows or to duplicate rows Modifying the Arrangement of the Acquisition Sequence You can modify the arrangement of the Acquisition Sequence by using shortcut menu commands to change the sorting order of the samples or to change the columns that are displayed in the Sequence table Right click in the Sequence table to display the shortcut menu Refer to the online Help for information on each of the commands In addition you can rearrange the order of the Acquisition Sequence by clicking on and dragging a row to move it To do this click on the row number in the left most column of the Sequence table and continue to hold down the mouse button Then drag the selected row up or down in the table A red horizontal line appe
117. ose File gt Import gt Processing Sequence gt Copy From Acquisition Sequence to copy the Acquisition Sequence from the current workbook into the Processing Sequence You can also right click anywhere within the Create Sequence Review and Edit page and choose Import Processing Sequence gt Copy From Acquisition Sequence from the shortcut menu to copy the Acquisition Sequence e By using the Acquisition Sequence History in the Create Sequence page If you have already acquired data in the workbook the Acquisition Sequence or Sequences that was used is listed in the Acquisition Sequence History list You can copy an Acquisition Sequence by selecting a sequence from the list and then dragging and dropping it into the Processing Sequence You can hold down the CTRL key to select multiple sequences If needed you can edit an imported or copied sequence Refer to the next topic Further Information Editing the Processing Sequence 96 LCquan User Guide Thermo Scientific 5 Creating a Processing Sequence Further Information Editing the Processing Sequence Further Information Editing the Processing Sequence You can use the Create Sequence Review and Edit page to alter the Processing Sequence by removing or adding raw sample files changing sample types and modifying the arrangement of the sequence as follows e Removing raw files from the Processing Sequence To select the row in the sequence that you want to delete
118. pe Response ISTD Response Response Ratio Specified Conc i tisone Explore 1 steroids02 Standard Method Settings 13775248 2435216 5657 5 000 Eenmaa Quantitate 2 steroids03 Standard Method Settings 7031020 2314238 3 038 2 500 progesterone g steroids04 Standard Method Settings 3666547 2595302 1 413 1 250 4 steroids05 Standard Method Settings 1782495 2403939 074 0 630 5 steroids13 Standard Method Settings 13449804 2361619 5 695 5 000 6 samplea Unknown Method Settings 13889320 2382340 5 830 NA g sampleb Unknown Method Settings 6882603 2370762 2 903 NA 8 9 sampled Unknown Method Settings 1899556 2391928 0 794 NA 10 samplee Unknown Method Settings 3575212 2463101 1 452 NA 11 steroids14 Standard Method Settings 7160845 2394666 2 990 2 500 12 steroids15 Standard Method Settings 3574151 2567027 1 392 1 250 13 ina 4 gt al Standards OCs Blanks Unknowns lt 3 i E 4 gt amp Selected Chromatogram v 4 gt amp ISTD Chromatogram v 4 gt amp Calibration Curve i steroidst6 TIC SM 7 PT 0 29 1 04 NL 148E7 F c Full m2 363 30 40 00 150 00 375 00 RT 0 67 Rette Ineas ty 043 0 50 o3 os os o6 o7 os Time min 09 steroidst TIC SM 7 AT 1 60 2 35 NL 3 59E5 F c Full ms 303 30 40 00 100 00 310 00 a RT 1 37 Rette heast Time min Area Ratio 2 25 24 22 23 Y 0 0293758 1 141
119. peak deoxycorticosterone in the multipeak plot LCquan displays the product ion mass spectrum at the maximum intensity 6 Record the mass to charge ratios of the first several ions of highest intensity in the deoxycorticosterone mass spectrum 7 Click on the third chromatogram peak methyltestosterone in the multipeak plot LCquan displays the product ion mass spectrum at the maximum intensity 8 Record the mass to charge ratios of the first several ions of highest intensity in the methyltestosterone mass spectrum 9 Click on the fourth chromatogram peak progesterone in the multipeak plot LCquan displays the product ion mass spectrum at the maximum intensity 10 Record the mass to charge ratios of the first several ions of highest intensity in the progesterone mass spectrum The three most intense product ions for the steroids are listed in Table 4 Table 4 Mass to charge ratios of the three most intense product ions of the steroids mass spectra hydrocortisone deoxycorticosterone methyltestosterone progesterone 309 2 253 1 285 1 297 1 267 2 295 2 267 3 279 2 327 1 109 0 227 2 255 0 You now have enough information to create quan components Go on to the next topic Creating a Quan Component from the Peak List 60 LCquan User Guide Thermo Scientific 3 Exploring Your Data Creating a Quan Component from the Peak List Creating a Quan Component from the Peak List This topic demonstrates how to create a quan
120. play buttons in the toolbar or the commands in the Zoom menu to zoom in or out along the axes of the spectrum You can also click and drag with the mouse in the Spectrum pane to zoom in Figure 23 Chromatogram pane and Spectrum pane showing a selected scan NL 2 11E5 F c SRM ms2 463 40 23 00 423 30 425 30 2 i 0 33 0 33 123 187 219 2 45 3 27 3 62 63 5 36 5 76 6 25 6 48 3 0 35 4 0 Time min Data file drugx_03 Number of traces 3 Relatie Intensity 423 6 423 6 424 0 424 2 424 8 425 0 Processing Data While Continuing to Acquire Data While you are acquiring data you can enter the Quantitate view to process the data already acquired If you have not yet defined a Processing Method or a Processing Sequence you need to complete these steps first Refer to Chapter 4 Creating a Processing Method and Chapter 5 Creating a Processing Sequence To enter the Quantitate view click on the Quantitate _Quantitate button in the viewbar You can choose to use the defined Processing Sequence or you can replace the defined Processing Sequence with a shortened sequence that is based on the data already acquired You can also specify whether the current Processing Sequence is updated with the most recent list of samples acquired From either the Acquisition view or the Quantitate view choose Options gt Acquisition Sequence Info to open the Acquisition Sequence dialog box Select the Auto
121. quisition view you can choose File gt Import Acquisition Sequence gt From File to display the Import Sequence File dialog box This dialog box allows you to import a sequence from an individual legacy file sld ipp or csv file or from an LCquan file qz file Thermo Scientific 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View You can also right click anywhere within the page and choose Import Acquisition Sequence gt From File from the shortcut menu to display the Import Sequence File dialog box e By dragging and dropping sequences from the Acquisition Sequence History pane The Acquisition Sequence History lists every Acquisition Sequence that was submitted to the acquisition queue in the life of the workbook You can select one or more of these sequences and then drag and drop them into the Sequence table Hold down the CTRL key to select multiple sequences e By copying the Processing Sequence if one already exists in the current workbook From the Setup Sequence page of the Acquisition view you can choose File gt Import Acquisition Sequence gt Copy From Processing Sequence to copy the Processing Sequence from the current workbook into the Acquisition Sequence You can also right click anywhere within the page and choose Import Acquisition Sequence gt Copy From Processing Sequence from the shortcut menu to copy the Processing Sequence Editing an Acquisition Sequence
122. r Guide H hydrocortisone calibration curve figure 106 ICIS peak detection 77 Identify Components page 74 importing Acquisition Sequence 38 Instrument Methods 27 Processing Method 85 Processing Sequence 95 raw files 24 injection volume autosampler 39 Instrument Methods creating 26 defined 25 importing 27 Instrument Setup view illustrated 25 opening 24 overview 25 Instrument Status 47 integrating peaks manually 110 integration markers 79 internal standards ISTDs defined 8 using for quantitation 8 ion ratio confirmation IRC description 83 hydrocortisone example 83 ion ratio confirmation IRC chromatogram 83 ISTD Chromatogram pane 102 L liquid chromatography LC using for analyte separation 3 variables for 3 locking plots for comparison 65 locking the workbook 121 locks against raw file change 65 lower quantitation limit 6 manual peak integration 111 manuals vii mass spectrometry analyte detection using 4 methyltestosterone internal standard calibration parameters 75 naming 75 Thermo Scientific peak detection and integration parameters 78 modifiers volatile 4 New Acquisition Wizard 30 New Method Wizard 72 New Processing Sequence Wizard 88 New Study or Workbook Wizard 23 0 overview of LCquan 10 18 P peak detection type Avalon 82 Genesis 82 ICIS 82 Peak Information dialog box illustrated 116 peak lists auto generated names 56 creating quan components 61 deoxycorticosterone
123. r modify the calibration levels and QC levels 1 Right click in the Sequence table and choose Standard and QC Levels from the shortcut menu to open the Acquisition Levels dialog box 2 Modify the Calibration Levels table as shown in Table 2 and Figure 18 Table 2 Calibration levels Calibration Level Amount ng cal01 0 63 cal02 1 25 cal03 2 50 cal04 5 00 cal05 10 00 Figure 17 Acquisition Levels dialog box with initial settings Acquisition Levels Components Levels component attevet ComponentO2 ComponentO3 4 Componento4 36 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View 3 Click on row 6 in the Calibration Levels table 4 Right click on the Calibration Levels table and choose Copy Current Component Levels to all Target Compounds from the shortcut menu 5 Change the component names by selecting the existing name in the Components table and then typing the new name a Change Componentl to methyltestosterone b Change Component2 to deoxycorticosterone c Change Component3 to progesterone d Change Component4 to hydrocortisone e Click on Row 5 in the Components table 6 Click on hydrocortisone in the Component List and change the cal05 level to 12 The Acquisition Levels dialog box should look like Figure 19 7 Click on OK Figure 18 Acquisition Levels dialog box showing components and calibra
124. race in the Spectrum pane Many of these options also appear in a shortcut menu that can be accessed by right clicking in the Chromatogram pane The elements of the User Defined Traces group box allow you to specify the settings for up to ten chromatograms For more detailed information about using the Realtime Display Settings dialog box refer to the online Help While LCquan is acquiring data you can also display an existing raw file in the Chromatogram pane using the current set of filters and settings LCquan continues to acquire data while displaying a raw file but the screen is in pause mode To display a raw file right click in the Chromatogram pane and choose Display Raw File from the shortcut menu The Select Rawfile dialog box appears from which you can choose the appropriate raw file You can also display a raw file by dragging it from the WindoW Explorer and dropping it into the Chromatogram pane or by dragging an older sample from the Acquisition Sequence History and dropping it into the Chromatogram pane With any chromatogram you can use the display buttons in the toolbar or the items in the Zoom menu to zoom in or out along the axes You can also click and drag with the mouse in the Chromatogram pane to zoom in When you zoom in the trace does not expand if the data extends out of the pane Thermo Scientific 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View Figure 22 Realtime
125. rk the calibration standards The process of reviewing and reworking the calibration standards involves the following steps 1 Opening the Survey Page 2 Reviewing the calibration standards 3 Excluding a Calibration Standard from the Calibration Curve optional 4 Modifying the Peak Detection and Integration Settings optional 5 Integrating Peaks Manually optional Opening the Survey Page LCquan User Guide After you create the Processing Sequence you are ready to review the calibration curves The Create Sequence Review and Edit page should be displayed See Reviewing the Processing Sequence on page 94 To process the raw files and open the Survey page click on the Survey button in the viewbar See Figure 47 The Survey page includes clockwise from the top the Result List the Component List the Calibration Curve pane the ISTD Chromatogram pane and the Chromatogram pane The Result List contains the peak integration results It has three display tabs All Standards and QCs Clicking on the All tab displays the results for all of the standards and QCs The Result List also uses a system of color shading to indicate the status of individual samples Refer to Table 7 Thermo Scientific 6 Processing the Raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Figure 47 Survey page of the Quantitate view showing the results for hydrocortisone A Cquan Steroids Workbook
126. rmo Fisher Scientific Inc and a purchaser This document shall in no way govern or modify any Terms and Conditions of Sale which Terms and Conditions of Sale shall govern all conflicting information between the two documents Release history Revision A December 2006 Revision B February 2007 Revision C April 2007 Revision D August 2007 Minimum software requirements LCquan 2 5 6 Xcalibur 2 0 6 TSQ Quantum 1 4 LC Devices 2 0 2 Thermo PAL 1 0 Microsoft Windows XP Professional SP 2 Microsoft Office 2003 For Research Use Only Not regulated for medical or veterinary diagnostic use by U S Federal Drug Administration or other competent authorities Contents Pietate a rer ere er eer vii About This Gude i cces cient ae eee ed RO eee hie chee nad Oo vii Related Documentation secu cans n diaow nace dor aces Kewanee sabelsad vii Special NGUCES cite ranana aa enw dee eens Oe hee ee aes viii Contacting Us nc cdae cabana bie dew Sia ode Daa dae E viii Chapter Introduction accutcnctcacesedentenassdabdivecusteeenstiocsstabiaadaneses 1 About Quantitative Analysis sos ese Wed 8 hee MO oes Leeds ee 2 Considering the Variables of Quantitative Analysis by LC MS MS 3 Using LC for Analyte Separations idence h i obra Gres eb eab tea oes 3 Using MS MS for Analyte Detection ac cee se acann eee daew eae ales amp 4 Quantitative Analysis Techniques 64 0 2000cee deo cde denen ee eee a 5 Using External Standards for Quantitativ
127. rmo Scientific Thermo Scientific 6 Processing the Raw Files and Reviewing the Analytical Results Creating and Reviewing Reports 3 To specify reports using the XReport templates do the following In the XReport Report Selections table select the Use check box in an empty row In the Save Type column select the file format in which you want to save the report In the XReport Template column type the name of an XReport template or click the empty cell and then click W to browse for a template The standard report templates contain preselected methods and results summaries See Table 8 on page 119 Alternatively click Create Report Templates _ to create a custom report template Tip To preview reports with your data open a template and then choose Report gt Simulate Report For information about XReport refer to Getting Productive Designing and Generating Custom Reports with XReport To create additional reports with XReport repeat steps a through c Note If your administrator has set up secure reporting the Create XReport Templates button is unavailable and only the templates in the secure template folder are available The only output option is Save Only which saves the file in pdf format 4 In the Select Report Options area select the output option You can choose Print Only Save Only or Print and Save 5 Click on Create Reports to generate the specified reports Note When you save a report us
128. rocessing Sequence by using the Sequence page of the Quantitate view A Processing Sequence provides instructions to LCquan on how to process a batch of raw data files It consists of a list of sample data files and includes information on sample type and calibration or QC levels Contents Opening the Create Sequence Page and Using the New Processing Sequence Wizard Associating Raw Files with Sample Types Reviewing the Processing Sequence Further Information Additional Ways to Define the Processing Sequence Further Information Editing the Processing Sequence LCquan User Guide 87 5 Creating a Processing Sequence Opening the Create Sequence Page and Using the New Processing Sequence Wizard Opening the Create Sequence Page and Using the New Processing Sequence Wizard In this example you use the Create Sequence Page of the Quantitate view to create a new Processing Sequence When you click on the Sequence icon LCquan opens the New Processing Method Wizard unless you have previously created a Processing Sequence in which case LCquan opens the Create Sequence Review and Edit page To create a new Processing Sequence for the steroids example 1 Click on the Quantitate button and then the Sequence icon to open the New Processing Sequence Wizard See Figure 42 2 Click on Next on the Welcome page of the New Processing Sequence Wizard to continue 3 Select the Create New Sequence option button and click on Next
129. s The Explore view of LCquan is similar to the Qual view of Processing Setup in Xcalibur Some features of the Explore view are e You can generate quan components automatically by using the peak list function e Explore view displays the Acquisition Sequence History and allows you to select a sample from it e Explore view does not allow you to edit sequences but you can import several sequences to review and select samples from any of the imported sequences e Things you do in the Explore view are not part of the audit trail except for exporting quan components to the Quantitate view Opening a Raw File and Displaying the Chromatogram You open a raw file that is representative of your data set to determine suitable peak detection and integration parameters In this tutorial you use the steroids04 raw file In general you choose a raw file corresponding to a low concentration calibration standard To enter the Explore view and open the steroids04 raw file 1 Click on the Explore button to enter the Explore view LCquan displays the Explore Create Explore Methods page 2 Open the steroid04 raw file a Choose File gt Open Raw File to open the Select Raw File dialog box b Browse to the Rawfiles folder or the Xcalibur Examples Data folder c Select steroidsO4 raw and click Open LCquan imports the raw data for the steroids04 raw sample and displays the raw chromatogram in the Create Explore Methods page of the E
130. s The following LC parameters might be considered for optimum separation of compounds e Composition of stationary phase e Column diameter e Column length e Column usability over time e Solvent s or composition of mobile phase e Flow rates e Isocratic or gradient ramped solvent compositions Often the only measurable result of optical detectors that LC chemists can use to identify an analyte is the retention time at a specified wavelength Therefore to quantitate compounds accurately the compound peaks must be distinctly separated from each other in time By carefully optimizing the LC variables listed above compounds can be successfully separated and detected with an optical detector such as an ultraviolet UV or photo diode array PDA detector In contrast to optical detectors mass spectrometers can often successfully detect individual analytes even when two or more compounds coelute as a single chromatographic peak Coeluting compounds are ionized in the mass spectrometer and the parent or fragment ions specific to the analyte s are detected and measured for quantitative analysis Because the mass spectrometer detection is so specific the chromatographic resolution can be reduced and the run times can be shortened LCquan User Guide 3 1 Introduction Considering the Variables of Quantitative Analysis by LC MS MS 4 Using MS MS for Analyte Detection LCquan User Guide Chemists who work with a mass spectrometer inter
131. s example is C Xcalibur examples method steroids sld You can always edit the sequence after you import it Click on Next Go to step 12 5 Specify the raw file names e Base File Name steroids e Starting Number 1 Click on Next LCquan appends a three digit number 001 999 to the base file name so that each sample in a sequence has a unique identification 6 Specify the unknown sample information e Number of Samples 5 e Injections per sample 1 e Base Sample ID sample Click on Next LCquan generates sample IDs that uniquely identify the unknown samples in the sequence by appending numerical values to a base sample ID 7 Specify the autosampler settings e Initial Vial Position A 1 e Re Use Vial Positions for replicate samples e Injection Volume Obtained from Autosampler Method Click on Next Thermo Scientific LCquan User Guide 31 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View 8 Specify the calibration standard information e Add Standards Based on Automatically Generated Cal Levels e Number of Cal Levels 5 e Cal Level Base Name cal e Number of Injections Per Level 2 e Add Blanks e Fill in Sample ID for Standards Click on Next LCquan generates names for the levels by appending numerical values to the base name Note e You can use the calibration levels and component names from the Processing Method if one exists in the workbook Refer
132. showing the progesterone peak properties for the samplea raw file Peak Info progesterone Info Flags Spectrum Left mir 2 9 Left height 8282 17 Apex min Peak Height 1130803 88 Right min H Right height 16295 25 Area cts sec 11236031 47 ISTD Response 2382340 17 Baseline 10727 78 Response Ratio Expected RT min 3 18 Calculated Amount Base Peak m z 297 15 Signal to Noise Peak Type Method Creating and Reviewing Reports You use the Review Reports page of the Quantitate view to create and review reports describing the quantitative analysis results for your data To create and review reports for your steroids data 1 Click the Reports button in the viewbar The Review Reports page appears See Figure 56 2 To specify Microsoft Excel reports do the following a In the Excel Report Selections table select the Use check box in an empty row b In the Column Arrangement column select a column arrangement from the list Three column arrangements are available by default Quan Result Grid Excel Long Summary and Excel Short Summary See Table 8 on page 119 for a description of each report type c To create additional Excel Reports repeat steps a and b d To create Sample Style Excel Reports reports with information on one sample per page rather than the default one component per page select the Sample Style Excel Reports check box 116 LCquan User Guide The
133. sition by selecting one of the After Sequence Set System option buttons On Standby or Off 9 Click on OK to save the settings and close the dialog box LCquan places the selected samples at the end of the Acquisition Queue and opens the Acquisition view Status page For more information see the online Help available within the software Note If you acquire data LCquan requires that you save the workbook Acquisition View Status Page The Acquisition view Status page provides a real time display of the data acquisition The following subtopics provide information on this page e Overview e Using the Chromatogram Pane e Using the Spectrum Pane Thermo Scientific LCquan User Guide 45 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View Overview The Acquisition view Status page consists of a set of control buttons an information pane that contains the Run Manager Status and the Instrument Status the Acquisition Queue and the Acquisition Sequence History the Chromatogram pane and the Spectrum pane See Figure 21 Figure 21 Acquisition view Status page quan Steroids Workbook File Change Actions View Apps Options Help a E X PEELE amp Run Manager Acquiing Sequence C Mealbur 28Jun2002 WB_28Jun2002i T emp Acq Sample Name Working On Seq Row 42 Position A 14 Raw File C Xcalibur 28 un2002 WB_28 un2002 R awfiles SCI Inst
134. so right click anywhere within the Create Sequence Review and Edit page and choose Import Processing Sequence gt From File from the shortcut menu to display the Import Sequence File dialog box Thermo Scientific LCquan User Guide 95 5 Creating a Processing Sequence Further Information Additional Ways to Define the Processing Sequence Note The level names in the Processing Sequence must match those defined in the Processing Method e Ifthe calibration or quality control level names in the sequence are different from those in the Processing Method LCquan displays the Standard Level Names Association dialog box or the QC Level Names Association dialog box You can use these dialog boxes to associate the level names in the sequence with the level names in the Processing Method Ifthe imported sequence contains calibration or QC levels but the Processing Method does not LCquan displays a warning box to inform you It then displays the Sample Type Assignment dialog box You can select to change the sample type of these samples to unknown in the Processing Sequence or to discard the samples that have these levels in the Processing Sequence Copying the Acquisition Sequence If an Acquisition Sequence already exists in the current workbook you can use it to define the Processing Sequence in the following ways e By using menu commands from within the Quantitate view From the Create Sequence pages of the Quantitate view you can cho
135. sponse Area Origin Include Weighting Equal LCquan User Guide 75 4 Creating a Processing Method Specifying the Component Identification and Integration Parameters C Click on Apply to apply the settings See Figure 38 Figure 38 Create Method Define Calibration Settings page of the Quantitate view showing the settings for hydrocortisone H LCquan Steroids Workbook DEK File Zoom Yiew Apps Options Help 5 J H HEGEREE PR COOTGOO LOO Identification Calibration Cerren deoxycorticosterone Quantitate te e ee methyltestosterone Larget Compound progester methyltestosterone he Cal Level QC Level Test OlsTD Isotope 1 Target Units Calibration Curve Type eo aean Linear x Response y Weighting ISTD Amount a sete 1 000 ri Egual O Height Ox ISTD Units A me Origin O1 x 2 Ignore Oww O Force One O Include O12 Apply Cancel Save As Default Help e Add fe Delete O Rename doa Chromatogram vy aoa Spectrum kg steroids04 TIC RT 0 51 1 26 NL 1 06E6 steroids04 79 RT 0 89 NL 3 67E3 T c Full ms2 363 30 40 00 150 00 375 00 100 0 68 100 327 21 S o 3 a 3 W S Y Ss Relative Intensity a o 8 S Peretti ties Fen ii Ss 5 06 07 0 8 1412 116 09 10 14 12 Time min 30 Relat
136. ss chromatogram for hydrocortisone AN LE quan Steroids Workbook EDEK File Zoom View Apps Options Help GeHE EELS COOSCOOO it OO 7 Chromatagram Definition Genesis Peak Integration Peak Identification ne Quantitate hyh Detector Smoothing Peak Detection Algorithm methy ya stostarone MS wi Genesis v Advanced Highest Peak Min Peak Height S N Proseperone a O Nearest RT 3 0 Trace S N Threshold 0 5 MassRange v v ES n lon ratio confirmation Mass m z Valley Detection Enabled E Enabled lon ratio using Area Eiter K Ratio i c Full ms2 363 30 40 00 150 00 375 00 vw Constrain Peak Width co Retention Time Peak Ht 5 0 Expected min 0 70 Window sec 30 0 Tailing Factor 1 0 i ai Window Use as RT Ref View Width min 0 75 sinka Qualifier ion coelution C Adjust Using Absolute min Apply Cancel Save As Default Help Add Delete Rename 4 gt 8 Chromatogram vy dra Spectrum v steroids04 m z 267 21 267 21 309 16 309 16 SM 7 RT 0 30 1 05 NL 1 59E5 steroids04 90 RT 1 02 NL 1 57E3 F c Full ms2 363 30 40 00 150 00 375
137. st Recently Used Workbooks list displays up to eight workbooks you can open a workbook from this list by clicking on it Figure 9 Startup dialog box for LCquan LCquan 2 5 A New Era in Mass Spectrometry ELECTRON CORPORATION Last used workbook C Xcalibur QuanRoot Example Study Steroids Workbook Most recently used Workbooks Open Last Used Workbook iC Open an Existing Workbook Create a New Workbook Summary Info for C gt lt calibur QuanRoot Example Study Steraids Workbook Created Tuesday September 06 2005 9 39 42 AM Modified Friday November 11 2005 4 26 05 PM Last saved by USSJO builder Number of saves 4 C Open in Review Mode 22 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis of Steroids Data Creating a New Study and Workbook 2 Ensure that the Open in Review Mode check box is cleared 3 Click on the Create New Study or Workbook button The Welcome page of the New Study or Workbook Wizard appears See Figure 10 4 Click on Next to continue 5 Name the study and workbook folders a In the Study Name text box type Practice Study b In the Workbook Name text box type Steroids Workbook c Click on Next to continue Figure 10 New Study or Workbook Wizard Welcome page New Study on Workbook Wizard Welcome to the New Study or Workbook Setup Wizard LCq ua 2 5 This wizard helps you set up a new Study or Wor
138. sults page of the Quantitate view to review and rework samples of all types including unknowns and blanks The process of reviewing the unknowns and blanks involves the following steps e Opening the Review All Results Page e Reviewing the Unknowns If necessary you can rework the samples by modifying the peak detection and integration settings and by integrating peaks manually For calibration standards and QCs the Review All Results page functions exactly as the Survey page does Refer to Reviewing the Calibration Curve and QCs on page 100 Note The steroids data set does not contain blank samples Opening the Review All Results Page To process the raw files and open the Review All Results page click on the Review All button in the viewbar See Figure 53 The Review All Results page includes clockwise from the top the Result List the Component List the Calibration Curve pane the ISTD Chromatogram pane and the Chromatogram pane These elements function exactly as they do in the Survey page The only difference is that the Result List now has five display tabs All Standards QCs Blanks and Unknowns Clicking on the All tab displays the results for every sample in the sequence 112 LCquan User Guide Thermo Scientific 6 Processing the Raw Files and Reviewing the Analytical Results Figure 53 Review All Results page of the Quantitate view showing the results for the steroids data uan Steroids Workbook
139. t raw file against the Explore Method that you created in the Create Explore Methods page Three categories of sequences appear in the sample selector e Explore Sequence e Quantitate Sequence e Acquisition Sequence History Explore Sequences are sequences that you imported into Explore by using the Load button You can use the Remove or Rename button to delete or rename the selected sequence Quantitate Sequences are Processing Sequences that you previously created in the Quantitate view The Acquisition Sequence History is historical information about the sequences that were used to acquire the data in the current workbook Note Raw files use absolute paths If you move the workbook LCquan will not find the samples and will create an empty graph LCquan User Guide 63 3 Exploring Your Data Additional Features of the Review and Explore Results Page Figure 31 Sample selector Current Review Display Multi Peak View Rawfile soem B Explore Sequences W reserpinecali sid reserpine03 reserpine04 reserpine0S reserpine06 reserpine l Quantitate Sequence w Processing Sequence steroids02 steroids03 steroids04 steroids05 steroids13 steroids14 steroids15 steroids16 sampleA sampleB samplec sampleD sampleE Acquisition Sequence History 64 LCquan User Guide Thermo Scientific Locks Thermo Scientific 3 Exploring Your Data Additional Features of the Review and Explore Results Page You us
140. tains separate pages for each instrument that you have selected by using the Xcalibur Instrument Configuration window Note The Instrument Setup view is not available in LCquan for Citrix Figure 4 Instrument Setup view AN LC quan Steroids Workbook File Quantum View Apps Options Help aA 4 e8 e Quantitate A IRETS Scan Editor Syringe Pump Divert Valve Accurate Mass Method Summary Run Settings MS Acquire Time min 10 00 Segments Current Segment 1 gt To display a chromatogram here use Quantum Open Raw File j al T Lg EAIA 2 aur F Lp 1 o 4 2 3 4 Retention Time min 6 7 8 9 10 Segment 1 Settings Segment Time min 10 09 Tune Method C Xcalibur Methods ex TSQTune 3 Scan Events 1 Chrom Filter Peak Width s C 10 Collision Gas Pressure mTorr 10 Scan Event 1 a pe Polarity Full Scan SIM SRM Positive O Negative Scan Modes MS Mode QiMS OQMS MS MS Mode Parent O Product O Neutral Loss Data Type Centoid Profile Scan Parameters Source CID Scan Range Collision Energy V 10 First Mass m z 150 000 Last Mass m z 1500 000 Accurate Mass Mode Off v Scan Time s 1 00 8 Q1 Peak width FWHM 070 APPI Lamp Set Mass m z 1000 000 Q3 Peak Width
141. tandards tab or the All tab is selected at the bottom of the Result List For each standard that you want to exclude from the calibration curve click in the check box in the Exclude column of its row To include the standard again clear the check box e By using the Exclusion List Ensure that the target compound is selected in the Component List Right click in the Calibration Curve pane to display the shortcut menu Then choose Exclusion List from this menu to display the Calibration Exclusion List dialog box For each standard that you want to exclude from the calibration curve click in the Exclude column of its row The word Yes appears in the Exclude column Click on Apply to incorporate the change To include the standard again click on the word Yes and then click on Apply e By using the Calibration Curve pane Left click on the data point in the Calibration Curve pane that you want to exclude and then right click to display the shortcut menu Choose Exclude from this menu to exclude the point To include the standard again left click on the data point Then right click and choose Include from the shortcut menu When you exclude a calibration standard in any of the above ways LCquan highlights in dark pink its row in the Result List and displays the excluded data point as an empty box in the Calibration Curve pane See Figure 51 Thermo Scientific LCquan User Guide 107 6 Processing the Raw Files and Reviewing the Analyti
142. the data 52 06 creating the Processing Method 71 07 create the Processing Sequence 88 08 processing the raw files 100 09 reviewing the calibration standards 102 10 reviewing the unknowns 114 Acquisition Sequence edited 35 Acquisition Sequence initial 29 calibration levels table 34 calibration levels and associated raw files table 91 calibration levels assigning 36 37 41 file names table 34 introduction 18 most intense ions 60 peak detection and integration parameters 77 peak list 54 sample types table 34 study creating 22 description 16 Survey page illustrated 101 108 manual integration 111 opening 100 using 100 T templates Explore view 69 templates importing or exporting Explore view 69 toolbar buttons for zooming note 104 U unknowns 9 upper quantitation limit 6 user guides vii user labels changing 40 V validation Citrix 19 variables quantitation with internal standards 8 separation by LC 3 W workbook creating 22 30 description 16 locking 121 saving 121 Thermo Scientific Index X X XReport 116 Z zooming in Chromatogram pane 48 in Spectrum pane 50 toolbar buttons note 104 Thermo Scientific LCquan User Guide 133
143. tion levels for the steroids example Acquisition Levels Components component t coe amount m 1 methyttestosterone deoxycorticosterone progesterone hydocortisone ectevet amount wrest To continue with the steroids example go on to Chapter 3 Exploring Your Data For more information on acquiring data and Acquisition Sequences continue with the next topic Importing or Copying an Existing Sequence Thermo Scientific LCquan User Guide 37 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View 38 Importing or Copying an Existing Sequence LCquan User Guide In some cases instead of creating a new Acquisition Sequence it might be more convenient to use an existing sequence as your Acquisition Sequence You can do this by importing an existing Acquisition Sequence or by copying the Processing Sequence from your current workbook You can import or copy an existing sequence in the following ways e By using the New Study Wizard When you create a new workbook the New Study Wizard gives you the option of importing Instrument Methods Acquisition Sequences and processing parameters to initialize the new workbook You can select to import all of this data from an existing workbook or you can select to import an Acquisition Sequence from an individual legacy file s d ipp or csv file or from an LCquan file gz file If you import an Acquisiti
144. trument collectively referred to as the Instrument Method as well as any optional instrument settings to be used before or after LCquan runs a sequence of samples Startup Method and Shutdown Method respectively The parameters that are displayed in the Instrument Setup view depend on the instrument that you select in the viewbar on the left side of the view See Figure 12 Note e The Instrument Setup view is not available in LCquan for Citrix e For this example if you do not create an Acquisition Sequence you need to create a Processing Sequence later Figure 12 Instrument Setup view showing the default Instrument Method for TSO Quantum AN L quan Steroids Workbook File Quantum View Apps Options Help Hae a ee Shutdown Method SSE EIEN TOES volte BS SIS TET IDOE TEI SD IIIT ETI IID SDI L AIN lt Scan Editor Syringe Pump Divert Valve Accurate Mass Method Summary Run Settings MS Acquire Time min 10 00 Segments 1 Current Segment 1 To display a chromatogram here use Quantum Open Raw File a en e Se a AA o 4 z 3 4 Retention Time min 6 7 8 9 10 Segment 1 Settings Segment Time min 10 00 Tune Method C Xcalibur Methods ex TSQTune J Scan Events 1 Chrom Filter Peak Width s C 10 Collision Gas Pressure mT orr J 1 0 pease ir SRM Polarity Ful Scan ll Positive O Negative Scan Modes MSMod QiMS
145. ts See Chapter 4 Creating a Processing Method Building a Processing Sequence A Processing Sequence consists of a list of sample data files and includes information on sample type and calibration or QC level See Chapter 5 Creating a Processing Sequence Processing the raw files and reviewing the calibration and quantitative analysis results After LCquan processes the raw data file you can evaluate and rework the calibration standards by modifying the peak detection and integration settings You can also integrate peaks manually See Chapter 6 Processing the Raw Files and Reviewing the Analytical Results LCquan User Guide 17 1 Introduction Introduction to Quantitative Analysis with LCquan 8 Creating reports LCquan provides both standard and custom printed reports Standard reports contain preselected methods and results summaries Custom reports contain methods and results summaries that you select from a list of options See Chapter 6 Processing the Raw Files and Reviewing the Analytical Results Using This Manual 18 LCquan User Guide This manual contains a stepwise procedure for performing quantitative analysis on an example data set provided in the Xcalibur examples data folder of your Xcalibur data system This data was collected on a steroids mixture in the applications laboratories at Thermo Scientific by using LC MS MS techniques in the APCI ionization mode The steroids mixture
146. ts Page Figure 30 Renamed peak list a Peak List steroids04 Explore deoxycorticosterone 1 383 258693 100 000 100 000 6 Right click on the peak list and select Create Quan Components gt From Selected Peaks in List from the shortcut menu to export the deoxycorticosterone quan component to the Quantitate view Create Method page For additional information on the Review and Fxplore Results page go on to the next topic Additional Features of the Review and Explore Results Page To continue with the steroids example go on to Chapter 4 Creating a Processing Method Additional Features of the Review and Explore Results Page Earlier in this chapter we described some useful features of the Explore view For example you can create an Explore Method apply it to the raw data to create a peak list and then used the peak list to create components for the Processing Method The following are additional features of the Review and Explore Results page that are useful 62 LCquan User Guide Sample Selector Locks General Parameters Plot Status Plot Status Report Review Displays Exporting the Active Raw File to Qual Browser Thermo Scientific Sample Selector Thermo Scientific 3 Exploring Your Data Additional Features of the Review and Explore Results Page The sample selector allows you to easily select a raw file See Figure 31 Click on a raw file in the sample selector to process tha
147. tton in the viewbar If no Acquisition Sequence is defined in the workbook the New Acquisition Wizard appears See Figure 14 Tip If the New Acquisition Wizard does not appear right click on the Sequence table and choose New Acquisition Sequence Wizard from the shortcut menu Figure 14 New Acquisition Wizard Welcome page New Acquisition Wizard Welcome to the Acquisition Setup Wizard LCqua n 2 5 This wizard helps you setup an Acquisition Sequence and if requested a new Study Workbook A New Era in Mass Spectrometry 2 Click on Next to continue 3 Specify not to create a new workbook for the acquisition Ensure that the Create a New Workbook check box is cleared and click on Next Note If you want to create a new workbook select the Create a New Workbook check box and click on Next Then provide a name for the new study and workbook and click on Next 30 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View 4 Specify not to import an existing sequence into the workbook Ensure that the Base Sequence On a Previously Saved File check box is cleared and click on Next Note If you want to import a sequence select the Base Sequence On a Previously Saved File check box Then click on Browse and locate the sequence file file with extension s d ipp csv or the LCquan file gm that contains the sequence The sequence for the steroid
148. uan LCquan is a complete quantitative analysis software package that enables you to acquire data specifically for an analyte of interest to quantitate the results and to produce reports This section contains the following topics e Overview of LCquan e LCquan Folder Structure e Acquiring and Processing Data with LCquan e Using This Manual Overview of LCquan LCquan is divided into four main views Instrument Setup Acquisition Explore and Quantitate By using these views you can develop Instrument Methods acquire data explore data and process data files 10 LCquan User Guide Thermo Scientific 1 Introduction Introduction to Quantitative Analysis with LCquan Instrument Setup View The Instrument Setup view of LCquan see Figure 4 enables you to develop Instrument Methods for the instruments that you have selected by using the Xcalibur Instrument Configuration window An Instrument Method is a set of experimental parameters and operating settings for a specific instrument such as an autosampler LC pump or MS pump mass spectrometer divert valve or syringe pump Within the Instrument Setup view you can create new methods or modify existing methods You can also import previously collected data files to help you set up time segments and scan events based upon the data acquired In general the Instrument Setup view within LCquan functions like the Instrument Setup window of the Xcalibur Home Page The Instrument Setup view con
149. uantitate view flow diagram LCquan Quantitate Flow Diagram Select Import Existing Select Create New Import Quantitation Method option Create or Import Method option Calibration Method Options Page Dialog Box in New Method Method in New Method P 9 Wizard then Wizard then Click on click on Next click on Next Click on Next Next Select Raw File Dialog Box Click on Next then click on Finish Create Method Identify Components Page Click on Calibrate tab Create Method Define Calibration Settings Page Click on Sequence button in viewbar Select Import Existing Select Create New Import Sequence Sequence option Create or Import Sequence option Dialog Box Sequence Create Sequence Select Files Page in New Processing Sequence Wizard in New Processing Sequence Wizard Click on then click on Next then click on Next Next then Click on click on Next in New Processing Sequence Wizard then click on Next Select Import From Acquisition Sequence option Finish Create Sequence gt Review and Edit Page Survey Standards and QCs Review All Results Review Reports Click on Click on Click on Survey button Review All button Reports button in viewbar in viewbar in viewbar Review All Review Survey Page Results Page Reports Page Thermo Scientific LCquan User Guide 127 Index A acquiring data overv
150. ure 26 54 LCquan User Guide Thermo Scientific Figure 25 Explore view Create Explore Methods page showing the peak list for the steroids example 3 Exploring Your Data Generating a Peak List 6 L quan Steroids Workbook E E x Eile Zoom Yiew Apps Options Help Lis eeocgooogt oe Taeniioater o Identification Chromatogram Definition ICIS Peak Integration Limit peaks Acquisition Sequence History Detector Smoothing Peak Detection Algorithm Select top peaks MS e Icis Advanced E Enable Trace Select by area Baseline Window ul w a al O Select by height Mass m z Area Noise Factor 5 Peak Noise Factor 10 Num to select 4 Eiter s C Constrain Peak Width Rel peak height threshold v me Peak Ht h 50 Cl Enable Benge ink Tailing Factor 7 9 of highest peak 100 0 0 999 9 prion ier par Save As Defaut Chromatogram Peak List steroids04 TIC RT 0 01 3 79 NL 1 06E6 RT 0 68 Peak Hame Apex RT Area of Largest Area of Total Area A 100 res J 1 0 675 3621463 100 000 34 358 J I H 4 904 2 34151 2715440 74 982 25 763 J 3 1 983 2633421 TAAT 24 984 J 4 1 392 1569926
151. ve 2 In the Comment text box type an optional comment to add to the Acquisition Sequence 3 Specify the instruments that you want to run your samples a b C Click on Change Instruments to open the Change Instruments In Use dialog box Specify the instruments that you want to use and the start instrument for the current Acquisition Sequence by clicking in the In Use and Start Instrument columns Yes appears in the column to indicate that the instrument is selected Click on OK to save changes and close the dialog box 4 Specify whether or not to start the acquisition when the instruments are ready e Ifyou want LCquan to start the data acquisition when the instruments are ready select the Start When Ready check box e Ifyou want LCquan to place the Acquisition Sequence in the paused state after all of the devices are ready clear the Start When Ready check box You must manually start the acquisition by using the control buttons in the Acquisition view Status page 5 Specify the processing actions Note LCquan can automatically process the data after it is acquired However a Processing Method must already be defined and the acquisition component names must match the processing component names If you want LCquan to process the data after it is acquired select the Process After Acquisition check box Then if you want to have LCquan print Sample Reports and Summary Reports after the data is processed
152. w to optimize the collision energy Quantitative Analysis Techniques Thermo Scientific To carry out quantitative analysis you need to evaluate the instrument s response to known amounts of the target component Response is based on either the height of the chromatogram peak or more commonly the area under the peak s profile See Figure 1 In both cases the baseline of the detected peak must be taken into account Instrument response is generally measured with several samples commonly called standards or calibration standards These standards must cover a suitably wide range of concentrations or amounts and must bracket the range of expected concentrations in the unknown Responses to these standards are plotted in a graph called a calibration curve deally this curve should correspond to the equation of a straight line to ensure the highest degree of precision Fitting an equation to the calibration curve with a user chosen method for example a least squares regression provides a response factor a comparative measure of the response of the mass spectrometer to a component It is based on the amount of sample injected and the resulting peak area or peak height Thus the response factor gives an intuitive and quantitative measure of how responsive or sensitive the mass spectrometer is to a certain component 2Mass Spectrometry Principles and Applications de Hoffman E Charette J Stroobant V Wiley New York 1996 p 162
153. xample before you acquire data you can create the Processing Method and Processing Sequence and then use these to define the Acquisition Sequence l Developing an Instrument Method non Citrix environment only LCquan uses an Instrument Method to store a specific set of parameters used to operate the autosampler LC pump or MS pump mass spectrometer divert valve and syringe pump during analysis See Chapter 2 Preparing to Perform Quantitative Analysis of Steroids Data Building an Acquisition Sequence of samples non Citrix environment only An Acquisition Sequence defines each sample as a standard unknown QC or blank and identifies its position on an autosampler tray if appropriate See Chapter 2 Preparing to Perform Quantitative Analysis of Steroids Data Acquiring data non Citrix environment only LCquan allows you to run one sample or a series of samples from the current Acquisition Sequence See Chapter 2 Preparing to Perform Quantitative Analysis of Steroids Data Exploring your data The Explore view is a sandbox where you can open a raw data file and play with peak detection and integration parameters that you will use in the Quantitate view See Chapter 3 Exploring Your Data Creating a Processing Method LCquan uses a Processing Method to identify detect and integrate components in a chromatogram generate calibration curves quantify unknowns and produce repor
154. xperiments you might save time by importing an existing Processing Method into your current workbook instead of creating a new method You can then use the method as is or edit it if necessary You can import a Processing Method in the following ways By using the New Study Wizard When you create a new workbook the New Study Wizard gives you the option of importing Instrument Methods Acquisition Sequences and processing parameters to initialize the new workbook You can select to import all of this data from an existing workbook or you can select to import a Processing Method from an individual legacy file pmd file or from an LCquan file gz file e By using the New Method Wizard in the Quantitate view When you use the New Method Wizard in the Quantitate view you have the option of importing a previously saved Processing Method You can import a Processing Method from an individual legacy file pmd file or from an LCquan file gz file e By using the menu command from within the Quantitate view From the Create Method page of the Quantitate view you can choose File gt Import gt Processing Method to display the Import Quantitation Method dialog box This dialog box allows you to import a Processing Method from an individual legacy file pmd file or from an LCquan file gz file Thermo Scientific LCquan User Guide 85 Creating a Processing Sequence Thermo Scientific This chapter describes how to create a P
155. xplore view See Figure 24 Four peaks which correspond to the four steroids appear in the chromatogram at retention times of 0 68 1 39 1 98 and 3 15 min 52 LCquan User Guide Thermo Scientific 3 Exploring Your Data Opening a Raw File and Displaying the Chromatogram Figure 24 Explore view Create Explore Methods page showing the raw chromatogram for steroids04 Note that you must click on a peak in the chromatogram to see a proper mass spectrum SN L quan Steroids Workbook File Zoom View Apps Options Help Gard Fj 0009000 4 0 0 Identification Chromatogram Definition ICIS Peak Integration Detector Smoothing Peak Detection Algorithm MS ind Icis Advanced Trace TIC v K Baseline Window 40 Mass m z Area Noise Factor 5 Peak Noise Factor 10 Filter O Constrain Peak Width Peak Ht 5 0 Range min Tailing Factor fi 0 03999 pees AO Limit peaks E amp Acquisition Sequence History w AcqSequence 1 Select top peaks E Enable Select by area Select by height Num to select Rel peak height threshold O Enable of highest peak 100 1 dra steroidsO4 TIC RT 0 01 3 79 NL 1 06E6 lt Chromatogram 100 F Relative Intensity RT 0 68 25 3 0
156. ys the Spectrum pane below the Chromatogram pane When clicked on again it hides the Spectrum pane The Run Manager Status provides a summary of the acquisition progress It lists the sequence and sample information and the readback status of each configured instrument The Instrument Status provides the complete readback status of each configured instrument The Acquisition Queue displays all of the sequences that have been submitted for acquisition including those for other workbooks The sequences and their individual sequence rows are organized in a tree view control Each row of the tree view control contains a check box to indicate when it is selected or not selected While a sample is being acquired its icon has a green background After the sample has been acquired the background changes to blue and a circle with an X through it appears in the check box You can remove a sequence or sequence row that has not been acquired from the Acquisition Queue by first selecting it and then pressing DELETE However you cannot delete a sequence that another workbook or application submitted You can view specific information about an individual sample by right clicking on the row in the queue and selecting Properties from the shortcut menu To display the Acquisition Queue click on the Acquisition Queue tab Thermo Scientific LCquan User Guide 47 2 Preparing to Perform Quantitative Analysis of Steroids Data Reviewing the Acquisition View

LCquan 2.5.6 Quantitative Analysis User Guide

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