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Xcalibur 2.1.0–2.3.0 Quantitative Analysis User Guide Version E

1. Parameter Description Levels Select one of the available levels for the Standard sample whenever you change a QC Blank or Unknown sample type to a Standard sample type in the Quan Browser window Results grid view When you select a level from the Levels list the read only information in the lower list displays the other component names that are assigned to the selected level and the amount assigned to the selected level for each component Levels Table Name View the components that are assigned the same level as the one currently selected in the Levels list Amount View the Component Amount assigned to the level currently selected in the Levels list for the component name selected in the Name column 118 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Select Report Samples Dialog Box The Select Report Samples dialog box displays the samples in the current bracket or group in the Sample Choices box From the Sample Choices dialog box you can pick which samples are to be processed when sample reports are selected Use the SHIFT and CTRL keys to select multiple samples The Xcalibur data system remembers the selected samples for each bracket until the application is terminated or a new file is opened Table 35 Select Report Samples dialog box parameters Parameter Description Sample Choices Raw File View or change the raw files in the current bracket or current g
2. 2012 Thermo Fisher Scientific Inc All rights reserved Xcalibur and Accela are registered trademarks and LCQ BioWorks and LCquan are trademarks of Thermo Fisher Scientific Inc in the United States Microsoft Windows Windows Vista and Excel are registered trademarks of Microsoft Corporation in the United States and other countries Adobe Acrobat and Reader are registered trademarks of Adobe Systems Incorporated in the United States and other countries The following are registered trademarks in the United States and possibly other countries Agilent is a registered trademark of Agilent Technologies Inc Oracle is a registered trademark of Oracle Corporation and or its affiliates Waters is a registered trademark of Waters Corporation All other trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries Thermo Fisher Scientific Inc provides this document to its customers with a product purchase to use in the product operation This document is copyright protected and any reproduction of the whole or any part of this document is strictly prohibited except with the written authorization of Thermo Fisher Scientific Inc The contents of this document are subject to change without notice All technical information in this document is for reference purposes only System configurations and specifications in this document supersede all previous information received by the purchaser Thermo Fisher Scientific Inc m
3. ae hydrocortisone ee Detector JI Type Ms bd Plot Type Mass Range v1 v1 x Scan Filter c Full ms2 363 30 150 00 375 00 v Mass m z 267 1 309 1 Peak Detection Algorithm Keys Genesis Y m Retention Time Expected min 0 64 Window sec 30 00 View Width min 1 00 Cancel Apply Apply Toan Help 2 Select the type of trace and optional trace math operation stored in the processing method in the adjacent Plot Type lists Only certain combinations of trace types are possible For more information about valid trace types see Integration Page User Identification Settings Dialog Box on page 130 To apply a different scan filter select a new filter from the Scan Filter list select a new filter from the Scan Filter list and edit it or type a new scan filter command string in the box using the scan filter format 38 Quantitative Analysis User Guide Thermo Scientific Thermo Scientific 4 Working with Peaks Setting User Integration Settings 4 View or change the masses stored in the processing method This display area changes to accommodate the type of data required When a single mass range is required a single edit box displays the current value If two mass ranges are required as in the case of a trace defined as a Mass Range Mass Range or Base Peak Mass Range this box is replaced by two boxes in the case of Base Peak Mass Range this box is replac
4. gt 1 lt Zoom In X To show more detail zoom in on the X axis by a factor of two 2 gt Zoom Out X To show more detail zoom out on the X axis by a factor of two 2 from the center SS DisplayAll View all data on the X axis or all text in a report Reset Scaling To Full Scale Thermo Scientific To display the maximum amount of data reset the scaling of both the X and Y axis Quantitative Analysis User Guide 79 A Quan Browser Reference The Quan Browser Window Table 8 Quan Browser toolbar buttons Sheet 3 of 3 Button Noise Description M Manual Noise Region a Specify a noise region manually Click kai and drag the cursor horizontally across the region of the chromatogram to be the noise region The data system marks the region with a red baseline The application calculates noise based on the data points you select It uses all selected data points as noise points and calculates noise based on those points You can select the noise region from an individual trace or different noise regions from multiple traces Open a raw file and select a chromatogram to make this button active Delete Manual Noise Region Remove a designated manual noise region Click X and drag the cursor over the region that was previously selected as the noise region Release the mouse button to delete the noise region Options View Stds And QCs View all components or just standards and quality control
5. After the application verifies that the files exist and can be opened the View Sample Types dialog box opens see Figure 5 Figure 5 View Sample Types dialog box View Sample Types Viewing Options Show Standard and GC sample types C Show All sample types l Don t ask again The two options provided in the View Sample Types dialog box determine how the Result Grid is configured at startup Thermo Scientific Quantitative Analysis User Guide 15 2 Quan Browser Overview Starting Quan Browser 3 Select one of these options e To display only Standards and QCs in the Quan Browser Grid view select the Show Standard and QC sample types option Blanks and Unknowns do not display Click either the Standards or QCs tab e To display Standards QCs Blanks and Unknowns in the Quan Browser Grid view select the Show All sample types option Click one of the following tabs All Standards QCs Blanks or Unknowns The View Sample Types dialog box includes a Don t ask again check box When you select this check box the dialog box is not displayed when you start subsequent sessions in Quan Browser and the current selection becomes the default Note To make this and all other Don t Ask Again type dialog boxes active choose Options gt Enable Warnings 4 To start the session click OK Quan Browser loads the specified sequence or file and configures the Results Grid using your selected viewing option 16 Quantitativ
6. Difference RSD Area Height Area Height Ratio Exclude File Name Integration Type Level Name Peak Status Sample ID Sample Type or Acquisition Date You can select and sort with any of these sort options even if the corresponding column is not currently displayed For example you can sort by sample type even if you have selected the Sample Name check box in the Result List Column Hiding dialog box Sort in descending order Sort the second criterion in descending reverse order If you do not select this check box the sort is in ascending order Third Order Base the third sort order of the Results grid view on any of the following column headings or file properties lt none gt Difference RSD Area Height Area Height Ratio Exclude File Name Integration Type Level Name Peak Status Sample ID Sample Type or Acquisition Date You can select and sort with any of these sort options even if the corresponding column is not currently displayed For example you can sort by sample type even if you have selected the Sample Name check box in the Result List Column Hiding dialog box Sort in descending order Sort the third criterion in descending reverse order If you do not select this check box the sort is in ascending order Button Save As Default 112 Quantitative Analysis User Guide Save your current selection of second and third sort orders as your default set The data system uses these s
7. The Component list displays all the components within the current bracket sorted by retention time To update the Chromatogram view and the Companion view with data for a specific component click the component name For more information about the Component list see Quan Browser Component List on page 84 Quantitative Analysis User Guide 17 2 Quan Browser Overview Quan Browser Window Overview Results Grid Bracket Group in Use Calibration File The Results grid is made up of the Bracket in Use list the Calibration File box and a sequence table Each row of the sequence table defines a result file and associated parameters Use the tabs at the bottom of the window to display all of the sequence samples or a subset of the sequence samples The Results grid is made up of these key areas e Bracket Group in Use e Calibration File e Results Grid View For bracketed sequences the Brackets in Use list shows the available brackets in sequential order The data system selects the first bracket in the list when the file is first loaded into Quan Browser and displays the samples within this bracket in the Results grid When you load an unbracketed sequence the samples are broken into logical groups The Groups in Use list shows the available groups Selecting a new bracket or group from the list refills the Results grid with the samples from the selected bracket or group The application updates all the other Views and dialog bo
8. i Quan Browser Overview This chapter describes how to use Quan Browser to review processed quantitation sequences It explains the properties and uses of each component within the Quan Browser window Qual Browser is described in the Xcalibur Qualitative Analysis User Guide Library Browser is described in the Xcalibur Creating and Searching Libraries User Guide Contents e Understanding How Quan Browser Works e Starting Quan Browser e Quan Browser Window Overview e Saving Changes Made in Quan Browser Understanding How Quan Browser Works Using the Quan Browser application you can step through a sequence of processed samples and review the results for each component in each sample The Xcalibur data system has the following quantitation features e Calibration Replicates e Named Calibration File e Brackets and Groups for Sequences Calibration Replicates Calibration replicates are multiple injections of the calibration mixture at the same calibration level or amount These standard samples all contain the same amount of target compound so they correspond to the same calibration level Choose replicates to include or exclude from the calibration curve by using the Calibration Curve companion view Thermo Scientific Quantitative Analysis User Guide 11 2 Quan Browser Overview Understanding How Quan Browser Works Named Calibration File After creating a sequence with the Bracket Type set to None specify a calibration file na
9. INCOS Noise Use a single pass algorithm to determine the noise level This value is used by the ICIS peak detection algorithm Repetitive Noise Use a multiple pass algorithm to determine the noise level This value is used by the ICIS peak detection algorithm In general this algorithm is more accurate in analyzing the noise than the INCOS noise algorithm but it takes longer RMS Min Peak Width Specify that the data system calculates noise as RMS By default the data system uses peak to peak for the noise calculation RMS is automatically selected if you determine the noise region manually Type the minimum number of scans required in a peak The valid range is 0 to 100 scans The default value is 3 scans This value is used by the ICIS peak detection algorithm Multiplet Type the minimum separation in scans between the apexes of two potential peaks This is a Resolution criteria to determine if two peaks are resolved The valid range is 1 to 500 scans The default value is 10 scans This value is used by the ICIS peak detection algorithm Area Tail Type the number of scans past the peak endpoint to use in averaging the intensity The valid Extension range is 0 to 100 scans The default value is 5 scans This value is used by the ICIS peak detection algorithm Area Scan Type the number of allowable scans on each side of the peak apex The valid range is 0 to 100 Window scans The default value of 0 scans specifies t
10. due to noise Saturation View the results of the Saturation test as Passed Failed or Not Tested This test indicates Thermo Scientific whether the detector was saturated during acquisition Quantitative Analysis User Guide 111 A Quan Browser Reference Quan Browser Dialog Boxes Quantitation Results Sorting Order Dialog Box Use the Quantitation Results Sorting Order dialog box to set the sort order for the samples in the Results grid view of the Quan Browser window The sort order defines the priority for each parameter used in the sort Table 31 Quantitation Results Sorting Order dialog box parameters Peak Information dialog box Parameter Sorting Description First Order Base the first sort order of the Results grid view on any of the following column headings or file properties lt none gt Difference RSD Area Height Area Height Ratio Exclude File Name Integration Type Level Name Peak Status Sample ID Sample Type or Acquisition Date By default the first order sort is set to the acquisition date of the file You can select and sort with any of these sort options even if the corresponding column is not currently displayed For example you can sort by sample type even if you have selected the Sample Name check box in the Result List Column Hiding dialog box Second Order Base the second sort order of the Results grid view on any of the following column headings or file properties lt none gt
11. files Save Create a new Quan Browser file with a xqn extension This file contains all the necessary information required to recreate the current browser session Save As Create a new Quan Browser file with xqn This file contains all the necessary information required to recreate the current browser session Gf oo Save All Update all result files with the current information Each result file comes from a results row in the Results grid Since each row can use method user or manual integration and the result file can contain only one method the Xcalibur data system uses the currently selected method for that row when creating the result file The system flags the embedded processing method as modified This means that each result file can potentially contain different embedded processing methods If read back into Quan Browser the system uses the first sample s embedded processing method for the entire bracket s method settings When read in it sets each sample that has a modified processing method in the User integration mode The operator must reintegrate and quantitate using a common method Reports Dialog Select and turn Report Templates on or off along with other options for sample reports and summary reports Print All Enabled Reports Print all active and currently selected sample reports as well as all active summary reports Print Enabled Sample Reports Print active and currently
12. list automatically To add a standard sample as a shared standard sample you must add it separately to each bracket The exclusion status of the replicates is independent for each bracket Even shared samples might be excluded in one bracket but not in another This is the only exception to a shared sample having identical settings 14 Quantitative Analysis User Guide Thermo Scientific 2 Quan Browser Overview Starting Quan Browser Starting Quan Browser To start the Quan Browser application 1 Do one of the following e On the Home Page Roadmap view click Be From the Instrument Setup window click x on the toolbar From the Home Page or Processing Setup windows choose GoTo gt Quan Browser At startup Quan Browser displays the Open dialog box so you can select an existing file If you do not want to select a file click Cancel in the Open dialog box to close Quan Browser The Quan Browser application supports these file types e Sequence files sld e Result files rst e Quan Browser files xqn Quan Browser handles result files as single entry sequences 2 Select a file in the Open dialog box When you select a sequence file the application checks that all the associated raw and result files are available When it encounters a problem with the sequence file the application provides information about the likely cause in a warning dialog box and prompts you to exit the application or select a different file
13. 131 A Quan Browser Reference Quan Browser Dialog Boxes Table 39 ICIS Integration page parameters User Identification Settings dialog box Sheet 2 of 2 Parameter Description Peak Ht View or adjust the percent of the total peak height 100 that a signal needs to be above the baseline before integration is turned on or off This box is active only when the Constrain Peak Width check box is selected The valid range is 0 0 to 100 0 To enter this height type the appropriate value in the Peak Ht box Tailing Factor View or adjust a factor that controls how the Xcalibur data system integrates the tail of a peak This tailing factor is the maximum ratio of the trailing edge to the leading side of a constrained peak This box is active only when the Constrain Peak Width check box is selected The valid range is 0 5 through 9 0 Avalon Integration Page Parameters The following table list the Avalon Integration page parameters Table 40 Avalon Integration page parameters User Identification Settings dialog box Sheet 1 of 4 Parameter Description Auto Calc Initial This button is active with the event list of the Avalon peak detection algorithm only if a raw file Events is open When you click the button Avalon automatically estimates the initial values for the detection of peaks based on the data in the current raw file and then displays those initial values in the event list Use this button to force Avalon to search for the
14. Box uit cjesicelie date scanebut eere 140 NGO E canis sce maaan tanks CaN E aaa dank ae 141 vi Quantitative Analysis User Guide Thermo Scientific ee i Preface This guide describes how to use the Thermo Xcalibur mass spectrometry data system to do quantitative analysis It describes how to review and rework your raw file data using the Xcalibur quantitative reviewing utility Thermo Xcalibur Quan Browser Contents e Related Documentation e Safety and Special Notices Contacting Us To provide us with comments about this document please click the link below Thank you in advance for your help SURVEY Before using this guide read your instrument s Getting Started Guide and the Xcalibur Getting Started Guide to become familiar with the basic features of the Xcalibur data system such as the Home Page and Instrument Setup Related Documentation Thermo Fisher Scientific provides these documents for the Xcalibur data system e Xcalibur Getting Started Quantitative Analysis e Acquisition and Processing User Guide e Quantitative Analysis User Guide e Qualitative Analysis User Guide e Creating and Searching Libraries User Guide e XReport User Guide e Help from within the software Thermo Scientific Quantitative Analysis User Guide vii Preface Safety and Special Notices Make sure you follow the precautionary statements presented in this guide The safety and other special notices appear in boxes Safety and speci
15. Factor is the number of points grouped together during peak detection It controls the bunching of chromatographic points during integration and does not affect the final area calculation of the peak The Bunch Factor must be an integer between 1 and 6 a high bunch factor groups peaks into clusters Area Threshold Controls the area cutoff Any peaks with a final area less than the area threshold is not detected This control is in units of area for the data P P Threshold The peak to peak resolution threshold controls how much peak overlap must be present before two or more adjacent peaks create a peak cluster Peak clusters have a baseline drop instead of valley to valley baselines This is specified as a percent of peak height overlap Negative Peaks Automatically resets after a negative peak has been found Tension Controls how closely the baseline should follow the overall shape of the chromatogram A lower tension traces the baseline to follow changes in the chromatogram more closely A high baseline tension follows the baseline less closely over longer time intervals Set in minutes Tangent Skim Using this event you can tangent skim any peak clusters By default it chooses the tallest peak in a cluster as the parent solvent You can also identify which peak in the cluster is the parent Tangent skim peaks are detected on either side or both sides of the parent peak Tangent skim automatically resets at the end of the peak clust
16. Failed or Not Tested This test indicates whether the peak is symmetrical about the apex Resolution View the results of the Resolution test as Passed Failed or Not Tested This test indicates whether multiple peaks are resolved If neither peak baseline endpoint is valley detected then the resolution passes Peak Width View the results of the Peak Width test as Passed Failed or Not Tested This test indicates whether the peak width is within specified limits Tailing View the results of the Tailing test as Passed Failed or Not Tested This test indicates whether the peak has tailing Column Overload View the results of the Column Overload test as Passed Failed or Not Tested This test indicates whether or not it is likely that the column was overloaded during acquisition This test is based on an analysis of the baseline and peak shape Baseline Clipping View the results of the Baseline Clipping test as Passed Failed or Not Tested This test indicates whether the baseline is clipped outside the peak This can occur if the chromatogram was started or terminated prematurely Signal to noise Ratio View the results of the Signal to noise test as Passed Failed or Not Tested This test indicates whether the minimum signal to noise ratio criteria are met Concave View the results of the Concave test as Passed Failed or Not Tested This test indicates whether the peak exhibits a concave depression local minimum
17. HANME data Ow methods Recent xdk E Desktop My Documents My Computer File name drugs_example2 XON My Network Save as type QuanBrowser xqn jna an xcalibur File or header is damaged Header Information 3 Type a name for the Xcalibur Quan file in the File Name box 4 To save the file and close the Save As box click Save 22 Quantitative Analysis User Guide Thermo Scientific Thermo Scientific 2 Quan Browser Overview Saving Changes Made in Quan Browser To save user integration settings to a processing method Apply the user integration settings to one sample or to all of the samples If you applied the user integration settings for a component peak to a specific sample and you want to save those settings to a processing method select the sample in the Results grid If you applied the user integration to all of the samples you do not need to select a specific sample in the Result grid Choose File gt Export Method The Save As dialog box opens Select the processing method that you want to modify or type a new name in the File Name box Click OK To export the information in the Results grid to an Excel spreadsheet Do one of the following e Choose File gt Export Data to Excel gt Export Short Excel Report e Choose File gt Export Data to Excel gt Export Long Excel Report The Microsoft Excel applicatio
18. ISTD option the ISTD box becomes active Go to step 5 To select an ISTD for the target compound option use the ISTD box to select an internal standard for the calibration To correct for Isotope Contributions click Isotope To define the ISTD for the ISTD option type values for the amount of the ISTD and the number of units 6 Repeat this procedure for all components Quantitative Analysis User Guide Thermo Scientific 5 Working with Calibration Settings Setting Curve Parameters Setting Curve Parameters Use the Curve page see Figure 24 to change the way the application calculates and plots the calibration curve from the data points To set calibration curve parameters for a target component 1 In the Quan Browser window open a sequence and select the target component that you want to modify in the components list 2 Open the Curve page of the Calibration Settings dialog box as follows a Display the Calibration Curve view in the Companion view by choosing View gt Set Companion View gt Show Calibration Curve b Right click the calibration curve and select Calibration Settings from the shortcut menu c Click the Curve tab Figure 24 Curve page Calibration Settings dialog box Calibration Settings Type Curve Levels Isotope Flags Calibration Curve N Weighting 7 Equal Origin CaN C Ignore C 1 Force CAN Include C 1 l a Response Area Units C Height
19. Plot View 000 cece eee eee 84 Quan Browser Spectrum Plot View oi oes ci geese esd ees tia dena ei ayes 86 Quan Browser Calibration Curve View 2 0 00 00 cece cece nes 87 Quan Browser Dialog Boxessi siti hin Pale ein Ga ween lala alge were bsatata i 89 AddSampl Dialog Box axni ice oA kate ak terete w ences aarp ee kote 90 Bracket Group In Use List 2 5 d b t cant satay dee andoatelon aie Aaphayes a ahtangied at ayeck 91 Cal Fxclusion List Dialog Box 4 tesco siete ete beeen tee eee 92 Calibration Settings Dialog Box 03 ws toss sewn dares eee ake aes 93 Curve Page Calibration Settings Dialog Box 0 000004 93 Flags Page Calibration Settings Dialog Box 00000 95 Isotope Page Calibration Settings Dialog Box 0 96 Levels Page Calibration Settings Dialog Box 0000004 98 Type Page Calibration Settings Dialog Box 0 000 99 Display Options Dialog Box in Quan Browser 00 000000 101 Masses Dialog Box x6 ant wate Ae nnee 101 Peak Information Dialog Box secu dace ede vee eb iewae ed Seeding 102 Chro Page Peak Information Dialog Box 000000 104 Flags Page Peak Information Dialog Box 00000 104 Info Page Peak Information Dialog Box 00000 107 Info More Info Page Peak Information Dialog Box 108 More Flags Page Peak Information Dialog Box 1
20. Right click in the Calibration Curve view except on a calibration point to display the shortcut menu for the view Calibration Settings Save Calibration File Exclusion List Show Spectrum Plot Reset Scaling Copy Graph Table 13 Calibration Curve view shortcut commands Command Calibration Settings Description Select values for the following parameters type curve levels isotope and flags These options appear when you display a valid calibration curve Save Calibration File Name and save the calibration file to the directory of your choice Exclusion List Select points in the calibration curve to include or exclude Show Spectrum Plot View the Spectrum Plot view in the lower right corner of the Quan Browser window To display the Spectrum Plot view choose one of these options e Choose View gt Set Companion View gt Show Spectrum Plot e Click Tl in the toolbar Right click the Calibration Curve view and select the Show Spectrum Plot command Reset Scaling Reset the plot scale to include the full peak in a normalized window Copy Graph Thermo Scientific Copy the Calibration Curve view to the clipboard so that you can transfer it to another open application such as Microsoft Word using the Paste command This operation is useful when you are writing a report and want to include the calibration curve Quantitative Analysis User Guide 87 A Quan Browser Re
21. Where TM impurity is an impurity compound in the target molecule reagent that elutes at the same time as the internal standard TM pure is the pure target compound To determine this ratio experimentally analyze the TM reagent using the method to be used for its quantitation Use the respective peak areas or heights to determine the ratio of impurity to pure compound The valid range is 0 00 to 100 00 percent To change the impurity ratio type a new value in the Contribution of Target Compound to Internal Standard box The data system uses this ratio as the value y in the following impurity correction expressions ISTD corr ISTD obs y TM obs 1 yx TM corr TM obs x ISTD obs 1 yx Where ISTD corr is the corrected amount of internal standard ISTD obs is the apparent amount of ISTD as measured by the data system at the retention time for ISTD pure This peak consists of ISTD corr TM impurity TM corr is the corrected amount of the target molecule TM obs is the apparent amount of TM as measured by the data system at the retention time for TM pure This amount consists of TM corr ISTD impurity See Contribution of Internal Standard to Target Compound box for a complete description of the variable x Quantitative Analysis User Guide 97 A Quan Browser Reference Quan Browser Dialog Boxes Levels Page Calibration Settings Dialog Box Use the Levels page of the Calib
22. and click Open The Add Sample dialog box opens with the parameter settings from the data file that you selected in the Results grid By default the Use Sample Name and Comment from Selected RawFile s check box is selected see Figure 12 Tip You cannot edit the following parameters in the Results grid Sample ID ISTD Corr Amt Dilution Factor Sample Name and Comment To enter different values for each sample add one sample at a time Note After you add samples to the Results grid you can change only the Sample Type the Level for a QC or Standard sample type and whether a calibration standard is included or excluded from the calibration curve for a component Figure 12 Add Sample dialog box la Add Sample o E Sample Type Level Standard x fi PG STD Sample ID 5 ISTD Corr Amt Dilution Factor 0 000 1 000 Settings from the sample selected in fi PG STD the Results grid 2 5 pg 10 mg Internal Standard e Cancel Help 4 Review the text entries and the Sample Type and Level selections Note The Comment and Sample Name boxes and the Use Sample Name and Comment from Selected Rawfile s check box are new features in the Xcalibur 2 3 data system Thermo Scientific Quantitative Analysis User Guide 29 3 Working with the Quan Browser Results Grid Adding and Removing Samples 5 For the Sample Name and Comments parameters do one of the following e To
23. and on the Info page for Candidate 2 and Candidate 3 of a spectrum search Forward View the forward threshold result for the current component read only Reverse View the reverse threshold result for the current component read only Match View the match threshold result for the current component read only Peak Info View the calculated peak characteristics for Candidate 2 or Candidate 3 of a spectrum search read only The Left Apex and Right peak boundaries in minutes and the Area and Height of the peak are given in units of counts This box on the More Info page does not appear for Candidate 1 of a spectrum search This information is provided on the More page that appears for Candidate 1 Left View the left extreme of the integration baseline for the current component in minutes read only Apex View the apex point in minutes of the integration baseline for the current component read only Right View the right extreme of the integration baseline for the current component in minutes read only Area View the area of the current component peak in units of count seconds read only Height View the height of the current component peak apex in units of counts read only 108 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes More Flags Page Peak Information Dialog Box Use the More Flags page of the Peak Information dialog box to review the foll
24. by Xcalibur for ion ratio confirmation Minimum Peak This parameter is available for the ICIS and Genesis algorithms View or change the peak Height signal to noise criteria that needs to be equaled or exceeded for the data system to use the Nearest RT Peak Identification criteria When identifying components the application ignores all chromatogram peaks that have signal to noise values that are less than the S N Threshold value The valid range is 0 0 all peaks through 999 0 128 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Table 37 Detection page parameters User Identification Settings dialog box Sheet 5 of 5 Parameter Description Buttons Apply Apply the current peak detection parameters to the selected component of the selected sample in the current sequence If one or more standard samples are changed the data system recalculates all quantitation parameters including peak areas and the calibration curve Apply To All Apply the current peak detection parameters to all samples that are currently displayed in the sequence For example if you select the Standards option the data system applies the current peak detection parameters only to the standards samples and not to any other samples If you select the All samples option the application applies the current peak detection parameters to all samples in the current sequence If one or more standard samples a
25. data Flags Page Peak Information Dialog Box Use the Flags page of the Peak Information dialog box to review the following peak properties for the current component Table 25 Flags page parameters Peak Information dialog box Sheet 1 of 3 Parameter Integration info Description Detected By View the method that the Xcalibur data system used to detect the peak Spectrum The application uses the user defined mass intensity pairs and applies a spectral matching algorithm to find the peak that contains the closest match to the comparison spectrum Up to 50 entries are allowed to define the comparison spectrum Highest Peak The application searches for the highest peak within the search window Nearest RT The application searches for the peak nearest to the expected retention time Left Edge Type View how the data system detected the left baseline edge of the current peak The data system displays one of the following peak baseline detection methods Edge Type Reported by Xcalibur Peak Criteria Met Baseline B The edge of the peak is at baseline level Valley V The edge of the peak is in a peak valley Manual M The edge of the peak has been adjusted manually Stripe S The edge of the peak reached the Constrain Peak Height Percent specified in the method Tail T The edge of the peak reached the Constrain Peak Height Trailing Factor limit before the Height Percent Tilt An error occurr
26. document that you have previously prepared Click Open The data system displays the full path and the name of the Report Template document with an xrt extension in the Report Template Name box Repeat steps 6 through 12 for the next row for each Report Template document that you want to use to print reports To select all of the sample reports you have selected for printing select the Include Sample Report check box at the bottom of the Reports dialog box 68 Quantitative Analysis User Guide Thermo Scientific Thermo Scientific 6 Viewing Results and Generating Reports Generating Reports 15 Click Select Samples The Select Report Samples dialog box opens The samples available from the sld file you opened in step 2 appear in the Sample Choices box 16 Select the samples for which you want to print reports For a group of contiguous continuous samples click the first of the sequence scroll until you see the last sample in the sequence and hold down the SHIFT key while you click The data system highlights the selected sequence For a group of non contiguous non continuous samples hold down the CTRL key while you click each sample to select or clear it The application highlights the selected samples 17 To copy the selected samples to the Selected Samples box click Add 18 To close the Select Report Samples dialog box click OK 19 From the Reports dialog box click Print Reports Prior to printing reports The dat
27. indicate that the report is enabled If you do not select the check box the cell remains blank when you click another cell Stds Thermo Scientific Specify whether or not the Standards Stds sample reports marked with Yes are processed using the report template that appears in the Report Template Name box of the same row To print a Standard sample report the data system must display Yes in the Enabled box in the same row For example if the Std box displays Yes and the Enable box in the same row displays Yes then the application prints the Std sample report However if the Enabled box in the row is clear then the application does not print the Std sample report When you click a Stds box a check box control appears If you select this check box and you click another cell the data system displays the word Yes to indicate that the report is enabled for Standard samples If it is not selected the cell remains blank when you click another cell Quantitative Analysis User Guide 113 A Quan Browser Reference Quan Browser Dialog Boxes Table 32 Reports dialog box parameters Sheet 2 of 4 Parameter QCs Description View whether or not the Quality Controls QCs sample reports marked with Yes are processed using the report template that appears in the Report Template Name box of the same row To print a Quality Controls sample report the data system must display Yes in the Enabled box in the same row For example if
28. is not closed View Menu Table 3 View menu commands Command Set Companion View Description Show Calibration Curve View the Calibration Curve view in the lower right corner of the Quan Browser window Show Spectrum Plot View the Spectrum Plot view in the lower right corner of the Quan Browser window Reports Dialog Specify the report template name to be used for sample reports or summary reports Toolbar View or hide the toolbar The toolbar appears if it was previously hidden or hides if it is currently displayed Status Bar View or hide the status bar The status bar appears if it was previously hidden or hides if it is currently displayed Show Large Toolbar View either the large or small Quan Browser toolbar The large toolbar is displayed when a check mark appears to the left of the command The small toolbar is displayed when the check mark to the left of the Show Large Toolbar command is cleared Customize Toolbar Thermo Scientific Drag any toolbar icon from this dialog box to any location on the Quan Browser toolbar and from the toolbar to this dialog box Quantitative Analysis User Guide 75 A Quan Browser Reference The Quan Browser Window Zoom Menu Table 4 Zoom menu commands Command Description 4 Zoom In Y To show more detail zoom in on the Y axis by a factor of two 2 from the current baseline iL Zoom Out Y To show more data zoom out on the Y axis b
29. laboratory The Calibration Curve companion view displays the calibration equation the goodness of fit parameter R and the weighting W 6 To adjust the calibration settings do the following a Right click the Calibration Curve companion view and choose Calibration Settings from the shortcut menu The Calibration Settings dialog box opens Figure 22 Calibration Settings dialog box Calibration Settings Type Curve Levels Isotope Flags Component Type Cc e ISTD methyltestosterone X 52 Quantitative Analysis User Guide Thermo Scientific 5 Working with Calibration Settings Modifying the Calibration Settings b Do the following To adjust the ISTD associated with the component modify the processing method in the Processing Setup window and batch reprocess the sequence in the Sequence Setup view For more information see Setting Component Types in the Processing Setup Window on page 54 To adjust the calibration equation weighting or units make new selections and entries on the Curve page For more information see Setting Curve Parameters on page 55 To view the calibration or QC levels click the Levels tab For more information refer to the Xcalibur Data Acquisitions and Processing User Guide To make corrections for isotope contributions to ISTD or Target components type new values on the Isotope page For more information see Changing the Isotope Percentage Value on p
30. left of the component names depending on whether or not the Xcalibur data system found the component based on the current detection criteria for the component Chromatogram plot view View the currently selected component chromatogram from the currently selected result file This view located in the lower left corner of the Quan Browser window displays a plot of relative abundance versus time in minutes for the selected component You can right click this view to display a shortcut menu to review and change peak detection and integration settings 72 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference The Quan Browser Window Table 1 Quan Browser workspace Sheet 2 of 2 Area Use Companion View Located to the right of the Chromatogram Plot view its companion in the lower right corner of the Quan Browser window In the companion view you can choose either the Spectrum Plot view or the Calibration Curve view Spectrum plot view View a plot of relative abundance Y axis versus mass to charge ratio for the selected component You can right click this view to display a shortcut menu to display the spectrum at the left edge apex and right edge of the chromatogram peak Also you can reset the scaling of the view Calibration curve view View a plot of area or area ratio Y axis versus concentration or amount X axis for the selected component You can right click this view to display a shortcut menu to revi
31. this option the calibration curve might or might not pass through the origin Force Specify that the calibration curve passes through the origin of the data point plot Include Include the origin as a single data point in the calculation of the calibration curve If you select this option the calibration curve might or might not pass through the origin Use the area or the height of the target compound peak to acquire the data used for the i calibration Area Use the area of the target compound peak to acquire the data used for the calibration Height Use the height of the target compound peak to acquire the data used for the calibration Thermo Scientific Quantitative Analysis User Guide 93 A Quan Browser Reference Quan Browser Dialog Boxes Table 18 Curve page parameters Calibration Settings dialog box Sheet 2 of 2 Parameter Weighting Description Equal Give all calibration data points equal weight during the least squares regression calculation of the calibration curve 1 X Specify a weight value of 1 X for all calibration data points during the least squares regression calculation of the calibration curve The data system assigns a weight value of the inverse of their quantity 1 X42 Specify a weight value of 1 X 2 for all calibration data points during the least squares regression calculation of the calibration curve The data system assigns a weight value of the inverse of the squa
32. valley detection type a value from 1 0 to 100 0 in the Valley S N box To apply the new peak detection criteria click OK To change the Peaks S N Cuttoff value type a value from 50 0 to 10000 0 in the Peak S N Cutoff box To apply the new peak detection parameter click OK For example if the signal to noise at the apex is 500 and the Peak S N Cutoff value is 200 the application defines the right and left edges of the peak when the S N reaches a value less than 200 To change how the baseline is drawn in the noise data type a value from 0 0 to 100 0 in the Baseline Noise Tolerance box To apply the new peak integration parameter click OK The higher the baseline noise tolerance value the higher the baseline is drawn through the noise data To change the minimum number of scans that the data system uses to calculate a baseline type a new value from 2 to 100 0 in the Min Number of Scans in Baseline box To apply the new baseline parameter click OK A larger number includes more data in determining an averaged baseline To change the number of background scans used to determine the background type a value from 1 to 100 in the Number of Background Scans box To apply the new baseline parameter click OK Click OK to save your changes 44 Quantitative Analysis User Guide Thermo Scientific 4 Working with Peaks Setting User Integration Settings Setting Flags Values To use the Flags page 1 From the User Ide
33. 09 More Info Page Peak Information Dialog Box 110 No Peak Page Peak Information Dialog Box 0 110 Spectrum Page Peak Information Dialog Box 0 110 Suitability Page Peak Information Dialog Box 111 Quantitation Results Sorting Order Dialog Box 0004 112 Reports Dialog Box and as x ep ene te Peles ha eee nate en 113 Result List Column Hiding Dialog Box ite cutce iin tiers dee es 117 Select L vel Dial g Box oyun hei ee iia E E ANE EE 118 Select Report Samples Dialog Box oe cc eta ayers uae ckatyeng hale dey a ionaneras ote 119 Quantitative Analysis User Guide v Contents User Identification Settings Dialog Boxi o0 oy naa solani tee at 120 Identification Page User Identification Settings Dialog Box 121 Detection Page User Identification Settings 00 125 Integration Page User Identification Settings Dialog Box 130 Genesis Integration Page Parameters 0 0 0 0 0 cece eee 130 ICIS Integration Page Parameters 6 06 eee cee 131 Avalon Integration Page Parameters ou ses aerate 132 Advanced Page User Identification Settings Dialog Box 136 Genesis Advanced Page Parameters icc ci venti ee wee ad Se 137 ICIS Advanced Page Parameters ig ised Senet tue aah hie Aude tis 138 Flags Page User Identification Settings Dialog Box 139 View Sample Types Dialog
34. 1 12 13 14 To print reports Choose GoTo gt Quan Browser or click el The Open dialog box opens To select a sequence select the name of the sequence file with an sld extension containing the samples you want to print reports from Click Open The View Sample Types dialog box opens Select the default Show All sample types option and click OK The data system displays the Results grid Component list Chromatogram Plot Spectrum Plot optional and Calibration Curve optional views Choose View gt Reports Dialog The Reports dialog box opens To make a row in the Sample Reports table active select the Enabled box in the first row Select the Enabled box again A check box appears in the box Select the check box When you click a different cell the word Yes appears in the Enabled box to indicate that the entire row of options is selected To select the report type that you want to print click one of the following boxes Stds QCs Unks or Other A check box appears Select the check box When you click a different cell the data system displays the word Yes in the report type cell to indicate that reports are to be printed for all selected samples of this sample type Repeat step 9 for all sample types for which you want printed reports Display the Open Report Template dialog box Double click a cell in the Report Template Name column In the Open Report Template dialog box select the
35. 3 Select a calibration curve type Use the Calibration Curve list to select Locally Weighted for the calibration curve type If you select Linear or Quadratic the Weighting box becomes active Go to Step 7 If you select any of the other curve types the Weighting box is not active Go to Step 8 4 To select calibration point weighting select from these options Equal 1 X 1 X 2 1 Y 1 Y42 or 1 s 2 This selection makes sure the data system applies the correct regression weighting method when it calculates the least squares regression calibration curve Thermo Scientific Quantitative Analysis User Guide 55 5 Working with Calibration Settings Setting Curve Parameters 5 Select how to treat the origin in the calibration curve calculation e To not include the origin in the calibration curve calculation select the Ignore option e To require that the calibration curve passes through the origin select the Force option e To include the origin as one data point select the Include option 6 To select the units to be displayed on graphs and reports type the required units label in the Units box 7 Select the response e To quantitate based on the integrated area of component peaks select the Area option Go to step 9 e To quantitate based on the calculated height of component peaks select the Height option Go to step 9 8 To select internal standard settings specify the units of the internal standard injected into
36. Box on page 119 e User Identification Settings Dialog Box on page 120 e View Sample Types Dialog Box on page 140 Thermo Scientific Quantitative Analysis User Guide 89 A Quan Browser Reference Quan Browser Dialog Boxes Add Sample Dialog Box Use the Add Sample dialog box to change information about the sample or samples that you are adding to the Results grid view For information about adding samples to the Results grid and changing the settings in the result files for the parameters listed below see Adding and Removing Samples on page 28 Note The Comment and Sample Name boxes and the Use Sample Name and Comment from Selected RawFile s check box are new features in the Xcalibur 2 3 data system Table 15 Add Sample dialog box parameters Parameter Description Sample Type Select a sample type for a new sample Level Select a level for a new sample Sample ID Enter the sample ID for a new sample Sample Name Enter the sample name for a new sample ISTD Corr Amt Enter the ISTD amount for a new sample Dilution Factor Enter the dilution factor for a new sample Use Sample Name and Comment from Selected RawFile s Comment 90 Quantitative Analysis User Guide To edit the Sample Name and Comments fields clear this check box To use the Sample Name and Comment embedded in the raw file select this check box Enter a comment about the new sample Thermo Scientific A
37. Exclusion List dialog box contains the name of the selected component Cal Exclusion List ComponentName The exclusion list displays all the replicates used in the current bracket or group and their exclusion status If there is a Yes in the exclude column then the Xcalibur data system excludes this replicate data point in the calibration curve plot If the column is blank then the Xcalibur system includes the replicate in the calibration curve plot To change the status of any replicate click the exclude column in the row containing the replicate data point This changes the value Click Apply to make the changes but keep the Exclusion List dialog visible Click OK to accept the changes and close the dialog box Click Cancel to abandon the changes and close the dialog box Table 17 Cal Exclusion List dialog box parameters Parameter Description Level View the read only calibration level names for the sample data points displayed in the Calibration Curve view Expected View the read only expected quantity values for the data points displayed in the Calibration Curve view Diff View the read only percent difference values for the data points displayed in the Calibration Curve view These values are the percentage difference between the calculated amount and the expected amount Exclude Click each row replicate in the Exclude column to turn Yes on and off Yes indicates that the data point is excluded from the data points d
38. Height Ratio Levels Units Retention Time Sample ID Specified Amount Calculated Amount Exclude Cancel Help To open the Result List Column Hiding dialog box Right click the Results grid and choose Columns from the shortcut menu Select the check box for a column heading to display it Clear the check box to hide the column To save your changes click OK Quantitative Analysis User Guide 31 3 Working with the Quan Browser Results Grid Changing the Sort Order Changing the Sort Order To change the sort order for entries in the Results grid 1 Right click the grid and choose Set Sorting Order from the shortcut menu The Quantitation Results Sorting Order dialog box opens Figure 14 Quantitation Results Sorting Order dialog box Quantitation Results Sorting Order Sorting First Order EER BF 1c v I Sort in descending order Second Order File Name X I Sort in descending order Third Order Level Name T I Sort in descending order Cancel Save As Default Help 2 To choose a heading for the primary sort of the Results grid select any of the following column headings or file properties e lt none gt e Area Height e Level Name e Difference e Area Height Ratio e Peak Status e RSD e Exclude e Sample ID e Acquisition Date e File Name e Sample Type e Integration Type By default the data system sets the first order sort to the acquisition date of the file Select and sort wit
39. Peaks There are two basic quantitation techniques e External standard ESTD quantitation e Internal standard ISTD quantitation The chosen method determines the calculation method both for the generation of the calibration curves and for subsequent quantitation This topic contains the following sections e Using External Standards e Using Internal Standards Using External Standards An external standard ESTD is a separate sample that contains a known amount of the target compound To perform an ESTD calibration prepare a set of standard solutions containing a known amount of the target compounds After you inject these solutions the data system analyzes the resulting chromatograms and constructs a calibration curve for each target compound by plotting the magnitude of the detector s response as a function of the amount of the target compound according to the following equation Response f Amount oal cal 7 Where f curve type Amount amount of calibration standard Response response for calibration standard The data system determines the amount of the target compounds in each unknown by comparing the magnitudes of their responses to the calibration curves see Figure 3 Use ESTDs if all compounds of interest can be assayed by using a single set of external standards This approach offers time and cost effective quantitation for applications using high precision autosamplers and traditional UV Vis detec
40. Quan Browser Reference Quan Browser Dialog Boxes Bracket Group In Use List Use this list to select the current bracket or group being browsed Select a new bracket or group from the list to refill the Results grid view with the samples from the selected bracket The Xcalibur data system updates all additional plots and dialog boxes automatically Table 16 Bracket Group In Use list parameters Parameter Bracket in use Description If a bracketed sequence is open the Bracket in use list is active and displays the available brackets numbered sequentially from one to the number of brackets in the sequence for example Bracket 1 Bracket 2 Bracket 3 and so on If the sequence contains multiple brackets the data system selects the first bracket in the list and displays the samples related to that bracket in the Results grid view If the sequence contains only one bracket then the list displays only one entry Bracket 1 Group in use Thermo Scientific If an unbracketed sequence is open the Group in use list is active and displays groups broken up logically For example Group 1 Group 2 Group 3 and so on Groups are separated by standards and are created using the following rules The data system scans the sequence file in chronological order until the first standard either Std Clear or Std Update is encountered The first standard begins a new group The application continues looking through the sequence adding all samples
41. See also Avalon Integration Page Parameters and ICIS Integration Page Parameters S N Threshold View or change the current signal to noise threshold for peak integration Peaks with signal to noise less than this value are not integrated Peaks with signal to noise greater than this value are integrated The valid range is 0 0 to 999 0 To change the current value type a new value in the S N Threshold box Valley Detection Use the Xcalibur valley detection approximation method to detect unresolved peaks This Enabled method drops a vertical line from the apex of the valley between unresolved peaks to the baseline The intersection of the vertical line and the baseline defines the end of the first peak and the beginning of the second peak To turn this method on select the Valley Detection check box To turn this method off ensure that the check box is clear Expected Width View the expected peak width parameter in seconds This controls the minimum width that a peak is expected to have if valley detection is enabled With valley detection enabled any valley points nearer than the expected width 2 to the top of the peak are ignored If a valley point is found outside the expected peak width the data system terminates the peak at that point The application always terminates a peak when the signal reaches the baseline independent of the value set for the expected peak width The valid range is 0 0 to 999 0 seconds To change the curren
42. Spectrum option for GC MS data e m z View the mass charge m z value for one peak in the reference spectrum The intensity for this m z value is given in the adjacent Intensity table box Use the other rows of the table to enter as many as 50 m z values The data system uses this data to identify the active component in the Find algorithm It displays this table only when you select the Spectrum option for GC MS data e Intensity Enter intensity data for one peak in the reference spectrum The m z value for this intensity is given in the adjacent m z Table box Use the other rows of the table to enter as many as 50 intensity values The data system uses this data to identify the active component in the Find algorithm It displays this table only when you select the Spectrum option for GC MS data Thresholds for the Spectrum option Forward Thermo Scientific Set a threshold value for Forward comparisons between the reference spectrum and candidates in the chromatogram A Forward search is a direct matching algorithm comparing unknowns against the reference spectrum in the peak identification table The match is scored on a scale of 0 to 999 A perfect match results in a score of 999 As a general guide 900 or greater is an excellent match 800 to 900 a good match 700 to 800 a fair match Less than 600 is a poor match Unknown spectra with many peaks tend to score lower than similar spectra with fewer peaks Quantitative Analysi
43. Update Expected Update the retention time specified in the processing method with the retention time that Retention Time the Xcalibur data system detected Update Component In Current Row command This command updates the retention time of the current component in the current row of the active sequence and recalculates the data for the row in the Results grid view Update All Components In Current Row command This command updates the retention time of all the components in the current row of the active sequence and recalculates the data for the row in the Results grid view Reset Scaling Thermo Scientific This command resets the plot scale to include the full peak in a normalized window Quantitative Analysis User Guide 85 A Quan Browser Reference The Quan Browser Window Quan Browser Spectrum Plot View When you select the Spectrum Plot view located in the lower right corner of the Quan Browser window as the companion plot the Spectrum Plot view displays a spectrum from the peak apex of the current chromatogram C Xcaliberiexamplesidataisteroids02 2128711996 11 20 31 AM steroids02 61 RT 0 68 AV 1 NL 4 18E5 F c Full ms2 363 30 cid 150 00 375 00 100 309 18 Spectrum at Peak Apex 80 60 40 20 0 Relative Abundance You can select a spectrum from a different time point in the chromatogram in two ways e Pin the Spectrum Plot view and then click a time point in the chromatogram e Rig
44. When the view is active pinned view scans from any part of the chromatogram by clicking the chromatogram Use the Spectrum Plot companion view to examine the identity of peaks and other features such as the background in the chromatogram For further analysis including library matching of spectra export data to Qual Browser using the Send to Qual Browser option in the Result list shortcut menu Initially the data system displays the spectrum corresponding to the scan at the current chromatogram s apex retention time If no peak is detected the application displays the expected retention time as defined by the processing method To display the Spectrum Plot companion view Do one of the following e Choose View gt Set Companion View gt Show Spectrum Plot e Right click the companion view and choose Show Spectrum Plot from the shortcut menu To display the spectrum for a specific time point in the current chromatogram 1 Make the Spectrum Plot view active by pinning the cell 2 In the Chromatogram view click the time point of interest To use the shortcut menu commands Right click the Spectrum Plot view and choose one of these commands from the shortcut menu e To display the spectrum at the apex retention time of the current chromatogram choose Spectrum at Peak Apex To display the spectrum at the left edge retention time of the current integration baseline choose Spectrum at Peak Left Edge e To display t
45. Xcalibur Data Acquisition and Processing User Guide Tip The report templates provided with the Xcalibur software are generic and might not produce the results you expect Preview a report in XReport before printing reports for an entire sequence For information about creating and changing reports refer to the XReport User Guide 7 After the Xcalibur data system processes the raw data files you can evaluate the peak detection settings the integration settings and the calibration curve for each component in Quan Browser As you evaluate the results of the processing method modify some of its parameters in Quan Browser If the processing method contains a report template print reports from Quan Browser Preview a report for a representative data file from the XReport reporting package To produce customized reports open a representative result file rst in the XReport reporting package and create a report template Once you are satisfied with the way a report displays your data add the report to the processing method if you have not already done so and batch process the sequence to generate printed reports Quantitative Analysis User Guide 3 1 Overview of Quantitative Analysis Integrating and Identifying Chromatographic Peaks Integrating and Identifying Chromatographic Peaks The Xcalibur data system integrates chromatograms to separate the chromatographic peaks from the baseline noise identify the beginning an
46. a system does the following Displays the Printer Activity icon at the bottom of the Quan Browser window Combines the data in the sample file you selected with the Report Template document you selected Displays the Save As dialog box Choose a file name for your report and click Save The data system adds the prefix Resolved to your report file name Adds the report printing task to the processing queue and then displays the Printing message box The data system returns you to the Quan Browser window after the reports print Quantitative Analysis User Guide 69 a B Quan Browser Reference Use the Quan Browser window to review sequence results for each component in each file You can quickly review component peak identification and integration criteria If you make any changes you can save the new results with an audit trail describing the reason for the change Use Quan Browser to interactively edit processing parameters and audit changes for individual result files It also creates new result files that contain the processing results for individual raw files These result files include a copy of the method used to generate the results Result files changed using Quan Browser do not affect the original processing method To edit processing methods refer to the Xcalibur Acquisition and Processing User Guide Contents e The Quan Browser Window e Quan Browser Dialog Boxes Thermo Scientific Quantitative Analysis Use
47. age 58 To change calibration and quantitation flag thresholds type new values on the Flags page For more information refer to the section Setting Calibration and Quantitation Flags in Chapter 4 of the Xcalibur Data Acquisitions and Processing User Guide c To apply any changes to the sequence click Apply 7 To exclude a point or sample from the calibration curve right click it and choose Exclude from the shortcut menu To include a previously excluded point right click it and select Include from the shortcut menu For both actions make sure your cursor is on the point For more information see Including or Excluding Data Points from the Calibration Curve on page 59 8 To exclude a level right click the Calibration Curve companion view and choose Exclusion List from the shortcut menu to open the Cal Exclusion List dialog box for the selected component e To exclude a level click the Exclude column adjacent to the level to be excluded For more information see Excluding Calibration Levels on page 57 e To restore an excluded level click the Exclude column adjacent to the level to be restored on the word Yes 9 To export the calibration settings with peak integration and detection parameters as a new processing method choose File gt Export Method Thermo Scientific Quantitative Analysis User Guide 53 5 Working with Calibration Settings Setting Component Types in the Processing Setup Window Set
48. ak at retention time of ISTD ISTD impurity ISTD pure e Ifyou have an impurity in your target molecule reagent that elutes at the same time as the ISTD molecule use the Target Compound to ISTD box to type the ratio TM impurityl TM pure To determine this ratio experimentally analyze the TM reagent using the method to be used for quantitation of the target compound Use the respective peak areas or heights to determine the ratio of impurity peak at the retention time of ISTD to pure compound peak at retention time of TM TM impurityl TM pure The data system corrects for the ISTD impurity TM impurity or both using the data you provide in Steps 1 and 2 and reports the corrected amounts of ISTD and TM 4 To save the settings and close the dialog box click OK Including or Excluding Data Points from the Calibration Curve To include or exclude data points 1 In the Quan Browser window open the sequence with the calibration that you want to modify as follows a Choose File gt Open The Open dialog box opens b In the Files of type list select sld Browse to find the correct file Select it and click Open The View Sample Types dialog box opens c Select the Show All sample types option and click OK The Results grid Component list Chromatogram Plot and either Spectrum Plot or Calibration Curve view open 2 To select the component for the calibration curve you want to view click a component in the Component list
49. akes no representations that this document is complete accurate or error free and assumes no responsibility and will not be liable for any errors omissions damage or loss that might result from any use of this document even if the information in the document is followed properly This document is not part of any sales contract between Thermo 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 January 2009 Revision B September 2010 Revision C January 2011 to reflect Microsoft Windows 7 compatibility Revision D May 2011 Revision E August 2012 Software version Thermo Xcalibur versions 2 1 0 to 2 3 0 For Research Use Only Not for use in diagnostic procedures B Contents Pr tate ee er Teer re eer eee vii Related Documentation ssu uu ds Mees oe be eas Kee eo eee Od vii Safety and Special Notes cduncosiue cin shee anced e nackte acts viii Contacting US er deca caddie vee eed tiaa dad hao daa ew Geo tae ean ix Chapter1 Overview of Quantitative Analysis ccc cece e eee teen eee 1 Acquiring and OQuattitatively Processing Data ccs cteavheneiabaaesdans 2 Integrating and Identifying Chromatographic Peaks 0000 4 Using External Standards Acusievnee siesta saws cen chee dene 6 Using Int
50. al 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 Highlights helpful information that can make a task easier viii Quantitative Analysis User Guide Thermo Scientific Preface Contacting Us There are several ways to contact Thermo Scientific for the information you need To contact Technical Support Phone 800 532 4752 Fax 561 688 8736 E mail us techsupport analyze thermofisher com Knowledge base www thermokb com Find software updates and utilities to download at mssupport thermo com To contact Customer Service for ordering information Phone 800 532 4752 Fax 561 688 8731 E mail us customer support analyze thermofisher com Web site www thermo com ms To copy manuals from the Internet Go to mssupport thermo com agree to the Terms and Conditions and then click Customer Manuals in the left margin of the window To suggest changes to documentation or to Help e Fill out a reader survey online at http www surveymonkey com s PQM6P62 e Send an e mail message to the Technical Publications Editor at techpubs lcms thermofisher com Thermo Scientific Quantitative Analysis User Guide ix ee B Overview of Quantitative Analysis The Xcalibur data system is a complete quantitative and qualitative analysi
51. aseline noise tolerance value the higher the baseline is drawn through the noise data The valid range is 0 0 to 100 0 Min Number Of View or adjust the minimum number of scans that the Xcalibur data system uses to Scans In Baseline calculate a baseline A larger number includes more data in determining an averaged baseline The valid range is 2 to 100 0 Number Of Background Scans Thermo Scientific View or adjust the number of background scans used to determine the background The valid range is 1 to 100 Quantitative Analysis User Guide 137 A Quan Browser Reference Quan Browser Dialog Boxes ICIS Advanced Page Parameters Table 42 ICIS Advanced page parameters User Identification Settings dialog box Parameter Manual Noise Region Description Specify the region of the chromatogram that the Xcalibur data system uses to determine noise You can click __ and drag the cursor horizontally across the region of the chromatogram that you want to select as the noise region or type the retention time RT in the RT Range box The data system marks the region with a red baseline RT Range Specify the retention time RT range The RT range should be within the chromatogram range You can click M and drag the cursor horizontally across the region of the chromatogram that you want to select as the noise region or type a value in the RT Range box The data system marks the region with a red baseline Noise Method
52. ata system displays 0 1 2 or 3 candidates in the Chromatogram Plot shortcut menu Candidates are ranked in the order of spectrum fit as 1 2 or 3 This shortcut menu shows a spectrum search result that found two matching candidates Method Settings User Settings s fanua Integration Show Peak Info Show Peak Info Candidate 2 User Peak Detection Settings Peak Labeling Manually Add Peak Set Peak to Not Found Status Update Expected Retention Time gt Reset Scaling Quantitative Analysis User Guide 49 4 Working with Peaks Reviewing Spectrum Search Results for GC MS Data 6 Choose a spectrum search option e Show Peak Info Not Found The data system did not find any peaks or could not find a spectrum match e Show Peak Info The data system found a spectrum match and this was the best spectrum match Candidate 1 Show Peak Info Candidate 2 The data system found a spectrum match and this was the second best spectrum match Show Peak Info Candidate 3 The data system found a spectrum match and this was the third best spectrum match 7 To review main component Candidate 1 results choose the Show Peak Info command the first of the two choices from the Chromatogram Plot shortcut menu The Xcalibur system opens the Peak Information dialog box with the following title Peak Information Component Spectrum Candidate and with the following pages Info More Info Flag
53. atio range the application truncates the range at 0 to avoid negative values e Absolute Specify that the target ratio tolerance values in the Window column of the qualifier ion table are absolute values For example if the target ratio is 50 and the Window parameter is 20 the expected target ion ratio range is 30 to 70 with the Relative option this would be 40 to 60 If the qualifier ion peak quantitation peak ratio is outside this range the ion ratio confirmation test has failed and the data system sets the IRC Flag to false If the qualifier ion peak quantitation peak ratio is within range the ion ratio confirmation test passes and the application sets the IRC Flag to true The response of all specified qualifier ions must be within the respective ratio ranges for IRC to succeed In assessing a target ion ratio range the data system truncates the range at 0 to avoid negative values Qualifier Qualifier Ion Coelution window Coelution e Prior to ion ratio confirmation the data system generates a mass chromatogram for each specified qualifier ion Each of these chromatograms must feature a peak matching that of the quantitation masses If the retention time of the qualifier ion peak apex lies outside of the Qualifier lon Coelution window centered on the quantitation peak the application rejects the quantitation peak e Quantitation peaks with matching qualifier ion peaks within the Coelution window are tested
54. best values of initial events that detect peaks in the data Any timed event in the event list is unchanged when you click this button Auto Calculate Initial Events determines initial values for the following events only Start Threshold End Threshold Area Threshold P P Resolution Threshold Bunch Factor Negative Peaks and Tension Additionally the user can specify timed events for these events in the same event list Smoothing Points View or adjust the number of points that the Xcalibur data system uses for chromatogram smoothing The valid range for smoothing points is from 3 to 15 The number of smoothing points must be odd To change the number of smoothing points type the new number of points in the Smoothing Points box 132 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Table 40 Avalon Integration page parameters User Identification Settings dialog box Sheet 2 of 4 Parameter Description Event List Event List To detect peaks Avalon uses the settings for initial events and user defined timed events in the event list To calculate values for initial events click Auto Calc Initial Events The event list in the Avalon Event List dialog box contains two hidden columns of information that are used by the algorithm and cannot be changed by the user Event OP Code and Value 2 There are seven initial entry integration events which are identified by t
55. ble Info Page Peak Information Dialog Box More Flags Page Peak Information Dialog Box Spectrum Page Peak Information Dialog Box Mote Info Page Peak Information Dialog Box Suitability Page Peak Information Dialog Box Spectrum Candidate Peak First Candidate Info Page Peak Information Dialog Box More Flags Page Peak Information Dialog Box More Info Page Peak Information Dialog Box Suitability Page Peak Information Dialog Box Flags Page Peak Information Dialog Box Chro Page Peak Information Dialog Box Flags Page Peak Information Dialog Box Spectrum Page Peak Information Dialog Box Spectrum Candidate Peak Second and Third Candidates More Info Page Peak Information Dialog Box Chro Page Peak Information Dialog Box Spectrum Page Peak Information Dialog Box Quantitative Analysis User Guide 103 A Quan Browser Reference Quan Browser Dialog Boxes Chro Page Peak Information Dialog Box The Chro page of the Peak Information dialog box is a read only page that displays the single mass chromatogram of the current qualifier ion The view centers on the retention time of the peak apex and has View Width specified in the Component Identification view of the Processing Setup window The mass of the qualifier ion is displayed in the title bar Qual Ion Mass xxx x You cannot change this display This page is only available for GC MS
56. bration Settings Select values for the following parameters type curve levels isotope and flags These options appear when you display a valid calibration curve Exclusion List Select points in the calibration curve plot to include or exclude Show Spectrum Plot View the Spectrum Plot view in the lower right corner of the Quan Browser window Reset Scaling Reset the plot scale to include the full peak in a normalized window Copy Graph Copy the calibration curve plot to the clipboard so that you can transfer it to another open application such as Microsoft Word using the Paste command This operation is useful when you are writing a report and want to include the calibration curve 88 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Quan Browser Dialog Boxes Quan Browser has the following dialog boxes e Add Sample Dialog Box on page 90 e Bracket Group In Use List on page 91 e Cal Exclusion List Dialog Box on page 92 e Calibration Settings Dialog Box on page 93 e Display Options Dialog Box in Quan Browser on page 101 e Masses Dialog Box on page 101 e Peak Information Dialog Box on page 102 e Quantitation Results Sorting Order Dialog Box on page 112 e Reports Dialog Box on page 113 e Result List Column Hiding Dialog Box on page 117 e Select Level Dialog Box on page 118 e Select Report Samples Dialog
57. bration curve is determined by the following equation Response RatiOTargetCal ISTD f Amount yyrgerCal Where Amounty rge Cal Amount of target compound in the calibration standards Response RatioTargetCal 1sTp ratio of the responses of the target compound to the internal standard compound in the calibration standard f equation of the calibration curve according to the selected fit type Ideally an ISTD is closely related to the target component in terms of its physical and chemical properties If the ISTD is used only to compensate for injection reproducibility or changes in the analyte solution volume it must possess a similar retention k to the target component but it does not need to be chemically similar to the target component 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 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 component Quantitative Analysis User Guide 9 ee
58. cified carry over threshold tolerance value to compare with the carry over threshold for the concentration of the quantified peak and determine if the carry over threshold is outside or within the specified tolerance Quantitative Analysis User Guide 109 A Quan Browser Reference Quan Browser Dialog Boxes More Info Page Peak Information Dialog Box Use the More Info page of the Peak Information dialog box to review the results of the following peak tests for the current component Table 29 More Info page parameters Peak Information dialog box Parameter Description lon Coelution Test Passed View whether the qualifier ion displayed in the title bar Qual Ion Mass xxx x passed or did not pass the Ion Coelution test If the check box is not selected no Ion Ratio test was performed and the Ion Ratio Test box is not displayed If the check box is selected the Ion Ratio test was performed and the Ion Ratio Test box on the Mass Info page is displayed at the bottom of the Mass Info page lon Ratio Test Passed View whether the qualifier ion displayed in the title bar Qual Ion Mass xxx x passed or did not pass the Ion Ratio test The Xcalibur data system does not display this box if the current qualifier ion did not pass the Ion Coelution test Target Ratio View the calculated Target Ratio Percentage that Xcalibur calculated during the Ion Ratio test read only The application does not display this box if
59. compound select the name in the Component list on the right side of the display Table 36 Identification page parameters User Identification Settings dialog box Sheet 1 of 4 Parameter Description Name This box displays a list of component names for the active processing method Plot Type These three lists display the type of trace and optional trace math operation that is stored in Thermo Scientific the processing method Only certain combinations of plot types are possible as shown in the following table 1st Plot Type Math Operation 2nd Plot Type Mass Range None n a Mass Range Mass Range TIC n a TIC Mass Range TIC Base Peak Base Peak None n a Base Peak Mass Range Analog 1 2 3 or 4 None n a Analog 1 2 3 or 4 di Digital 1 2 3 or 4 Digital 1 2 3 or 4 None n a Digital 1 2 3 or 4 H Digital 1 2 3 or 4 Quantitative Analysis User Guide 121 A Quan Browser Reference Quan Browser Dialog Boxes Table 36 Identification page parameters User Identification Settings dialog box Sheet 2 of 4 Parameter Description Filter This box displays the current scan filter for the active file of extension raw You can use a scan filter to specify that processing is to be applied to a subset of the scans in a raw file To apply a different scan filter select a new filter from the scan filter list most common method select a new filter from the list and edit the scan filter or type a new sca
60. d Baseline Clipping Not Tested Signalto noise Ratio Not Tested wi Concave Not Tested X Use the parameters on Suitability page to determine if the LC column is degrading and to identify suspicious peaks eluting at the same time as the target compound Suspicious peaks due to highly retained compounds from a previous injection tend to have a broader than expected peak profile Tailing peaks frequently indicate a degrading LC column There are three possible results for each test e Passed e Failed e Not Tested C kod To set up the system suitability criteria for a component peak 1 In the Processing Setup window open the processing method that you want to modify N Open the Quan view of the Processing Setup window KOS Click System Suitability tab The System Suitability page opens 4 For each component that you want to modify do the following a Inthe Components list select the component b Modify the settings on the System Suitability page c Click OK For more information about setting the system suitability parameters in the processing method for a quantitative analysis refer to the Xcalibur Data Acquisition and Processing User Guide 36 Quantitative Analysis User Guide Thermo Scientific 4 Working with Peaks Setting User Integration Settings Setting User Integration Settings Thermo Scientific When you first open a sequence in Quan Browser component identification the ap
61. d end of each peak identify the peak maxima and calculate the area or height of each peak see Figure 1 The data system uses one of the following three algorithms to detect and integrate the peaks in chromatograms Genesis ICIS or Avalon To integrate mass chromatograms use either the Genesis or the ICIS integration algorithm To integrate UV Vis and analog chromatograms use the Avalon integration algorithm Figure 1 Integrated chromatographic peak showing peak start and peak end markers RT 0 68 100 90 80 70 60 50 40 Relative abundance 30 Peak start Peak end 20 10 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 0 1 1 Time min For LC data the Xcalibur data system identifies peaks based on their retention times The retention time of a peak is the time that elapses between the injection of the sample and the detection of the peak maxima For GC data The application identifies peaks based on either their retention times or their mass spectra During a sequence run the retention times of chromatographic peaks can vary slightly As a result enter an appropriate retention time window for each peak in addition to its expected retention time A retention time window is a time range bracketing the discrete retention time setting The appropriate retention time window for a chromatographic peak depends on several factors including the width of the chromatographic peak and the specificity of the chromatographic method Due to band broad
62. d in the Summary Reports box Select Samples Select the samples for which you want to print reports Print Reports Print the reports for the samples you have selected 116 Quantitative Analysis User Guide Thermo Scientific Result List Column Hiding Dialog Box A Quan Browser Reference Quan Browser Dialog Boxes The Result List Column Hiding dialog box contains the list of possible columns that can be viewed in the Results grid Selecting an entry forces that column to be displayed and clearing the check box removes the column from the grid Table 33 Result List Column dialog box parameters Parameter Selected Columns Description These settings indicate whether or not to display a column in the Results grid The following columns are available Thermo Scientific File Name Sample Type Sample Name Integration Type Area Height ISTD Area Height Area Height Ratio Specified Amount Calculated Amount Percent Difference Percent RSD Peak Status Levels Units Retention Time Sample ID Exclude Quantitative Analysis User Guide 117 A Quan Browser Reference Quan Browser Dialog Boxes Select Level Dialog Box Note Whenever you change a QC Blank or Unknown sample type to a Standard sample type in the Quan Browser window Results grid view the Select Level dialog box opens so that you can select one of the available levels for the Standard sample Table 34 Select Level dialog box parameters
63. deds deh dteiielavienstatiownes 32 Thermo Scientific Quantitative Analysis User Guide iii Contents iv Chapter 4 Chapter 5 Chapter 6 Quantitative Analysis User Guide Working with Peaks ccc cece cece eee eee eee e eens 33 Viewing and Changing Peak Identification Detection and Integration 33 Setting the Chromatogram and Spectrum View Display Options 34 Viewing Peak Information a4 0 2 ates i oo wars Ohio wG Ae Ales nate 35 Setting User Integration Settings 0 0 0 ee ec eee eee ee 37 Setting Identification Values ssi Seausat Seen bib unune 38 Setting Detection Valtesirgay caGie iy ae ne een cel hoe Wands 40 Setting Genesis Integration Values occ tention weaker witha she 4 Setting Genesis Advanced Parameter Values 0 0000 eee eee 43 Setting Flags Valier soen cue dated te he etd t die Puli gion ih tabi ta 45 Integrating Chromatogram Peaks Manually 0 00020000 46 Using the Spectrum Plot Companion View 0 00 cee eee eens 48 Reviewing Spectrum Search Results for GC MS Data 0000 49 Working with Calibration Settings 0 0 cece cece eee eee eee 51 Modifying the Calibration Settings nav dee seaed tek ee eo 51 Setting Component Types in the Processing Setup Window 54 Setting Curve Parameters perint i re diyt E R E dao EE eee ede 55 Excluding Calibration Levels vec edeatios ure otdpatees uta ate g a as
64. dentification Settings dialog box click the Detection tab Figure 18 Detection page User Identification Settings dialog box User Identification Settings Nearest RT Min Pk Ht S N Cancel Apply Toan Help 2 To use the highest peak in the chromatogram for component identification select the Highest Peak option 3 To use the peak with the nearest retention time in the chromatogram for component identification select the Nearest RT option 4 To change the peak signal to noise criterion that needs to be equaled or exceeded for the data system to use the Nearest RT Peak Identification criterion type a value from 0 to 999 0 all peaks in the Min Pk Ht S N box For Component Identification purposes the application ignores all chromatogram peaks that have signal to noise values that are less than the S N Threshold value 5 Click OK to save your changes 40 Quantitative Analysis User Guide Thermo Scientific 4 Working with Peaks Setting User Integration Settings Setting Genesis Integration Values The Xcalibur data system applies the settings on the Integration page see Figure 19 during peak integration To set Genesis integration parameters 1 From the User Identification Settings dialog box click the Genesis Integration tab Figure 19 Genesis Integration page User Identification Settings dialog box User Identification Settings Identification Detection Genesis Integration Genesis A
65. detection 5 of quantitation 5 lower quantitation limit 5 manual peak integration 46 nonoverlapped bracket type 14 0 open bracket type 14 Options menu Quan Browser 76 overlapped bracket type 14 P Peak Detection Settings command User Identification Settings dialog box 37 Peak Information dialog box 35 102 peaks integrating manually in chromatogram 46 Print Reports button 66 processing methods saving 23 Q Qual Browser sending result file to 48 Quan Browser Cal Fxclusion List dialog box 57 Chromatogram view 19 component list 17 getting started 15 opening files 15 Peak Information dialog box 35 Quantitation Results Sorting Order dialog box 32 Reports dialog box 66 Result List Column Hiding dialog box 31 Select Report Samples dialog box 67 title bar 73 User Identification Settings dialog box 37 View Sample Types dialog box 15 window features 17 142 Quantitative Analysis User Guide Quan Browser views Calibration Curve view 87 Chromatogram Plot view 84 Component List view 84 Results grid view 81 Spectrum Plot view 86 quantitation limits 5 quantitation range 5 Quantitation Results Sorting Order dialog box 32 quantitative analysis definition 2 sources of error 7 using internal standards for 7 Replace Calibration command 18 replicates 11 Reports dialog box Select Samples button 67 reports generating 66 Reset Scaling command 48 result files saving 21 Result List Column Hiding dialog box 31 Re
66. displayed in the grid Summary Information View summary information for the current xqn file Change Dataset Name Select a dataset from a predefined list of names The text of this menu item might be different if the administrator chooses to use another name for a dataset For example this menu item might be Change Job Name Audit Trail Print Setup 74 Quantitative Analysis User Guide View all auditable events and changes made to data files in the current application Select a printer paper and page orientation Thermo Scientific A Quan Browser Reference The Quan Browser Window Table 2 File menu commands Sheet 2 of 2 Command Print Description Reports Dialog Select and turn Report Templates on and off along with other options for sample reports and summary reports All Enabled Reports Print all active and currently selected sample reports as well as all active summary reports Enable Sample Reports Print active and currently selected sample reports Enable Summary Reports Print all active summary reports Recently Used Files View the paths and names of the most recently used files located above the Exit command The data system displays both open and closed files Click a displayed file to load it If the selected file is closed the application reopens it Exit Close the Quan Browser window This option does not close any other Xcalibur windows The Home Page window
67. dvanced Flags Smoothing Points S N Threshold V Valley Detection Enabled Expected Width sec Peak Ht Tailing Factor Cancel Apply Apply To All Help 2 To specify the degree of data smoothing to be performed on the active component peak prior to peak detection and integration type any odd number between 1 no smoothing through 15 maximum smoothing in the Smoothing Points box 3 To specify the lowest signal to noise threshold for integrating peak type any value from 0 0 to 999 0 in the S N Threshold box The data system does not integrate peaks with signal to noise less than this value 4 To use the Xcalibur valley detection approximation method to detect unresolved peaks select the Valley Detection Enabled check box This method drops a vertical line from the apex of the valley between unresolved peaks to the baseline The intersection of the vertical line and the baseline defines the end of the first peak and the beginning of the second peak Thermo Scientific Quantitative Analysis User Guide m 4 Working with Peaks Setting User Integration Settings Specify the expected width of the peak in seconds 0 0 to 999 0 seconds If you selected Valley Detection the data system ignores any valley points nearer than the expected width 2 to the top of the peak If a valley point is found outside the expected peak width the application terminates the peak at that point It always terminates a peak when
68. e Analysis User Guide Thermo Scientific 2 Quan Browser Overview Quan Browser Window Overview Quan Browser Window Overview Figure 6 Title bar Menu bar once t 4 t t olole MIE 2 Toolbar Status bar The Quan Browser window see Figure 6 has the following features e Component List Results Grid e Chromatogram and Companion Views For more information about the Quan Browser window see Appendix A Quan Browser Reference Quan Browser window Results grid Component list Be steroid sld Bracket 1 View All Options GoTo Help Quan Browser File view Zoom Calibration File Embedded Calibratic n ai TRYS cket 1 deoxycorticosterone methyltestosterone progesterone Bracket in use Gi Integration Type steroids03 Standard Method Settings 3 steroids04 Standard Method Settings 4 steroids05 Standard Method Settings 5 steroids13 Standard Method Settings e steroids14 Standard Method Settings 7 steroids15 Standard Method Settings a steroids1 6 Standard Method Settings DENA Sans AQcs i Blanks Unknowns NL 5 01E5 6 g 100 miz 266 6 267 6 308 6 309 6 gt S F Full ms2 363 30 2 150 00 375 00 MS Genesis Eg 3 steroids02 9 a 50 E k 2 i 2 0 52 0 95 o os 19 o 2 4 6 8 10 12 Time min NUM 10 24 2004 5 45PM 4 Chromatogram view Companion view Component List Thermo Scientific
69. e Sort Order on page 32 8 To save your changes do the following e To save your edits to an Xcalibur Quan file extension xqn choose File gt Save The resulting Xcalibur Quan file extension xqn contains all the necessary information required to recreate the current Quan Browser session e To save your edits to an Xcalibur Quan file extension xqn and add information to the audit trail choose File gt Save As e To update and save the result files choose File gt Save All The application updates the result files with the new information and current time stamp 26 Quantitative Analysis User Guide Thermo Scientific 3 Working with the Quan Browser Results Grid Reviewing the Peak Status Column Reviewing the Peak Status Column Thermo Scientific If the sequence contains QC samples you can check the stability of the chromatographic method by viewing the results in the Peak Status column of the Result grid of the Quan Browser window If the calculated amount for the QC sample differs by more than the specified percentage in the Processing Method the peak status is listed as QC Failed KD I To check the stability of a sequence run that contains QC samples Open the sequence of acquired and processed data in the Quan Browser window Click the QCs tab In the Components list select a component View the results in the Peak Status column of the Result grid The three possible results for a QC sam
70. e difference between the calculated amount and the specified amount Relative Standard View the standard deviation of the difference between the calculated amount and the Deviation specified amount as expressed as a percentage of the specified value Peak Status View the peak status Low if the Difference is lt 0 High if the Difference is gt 0 Fail if the Difference is gt the QC fail percentage test value Level View the calibration or QC level of the sample To change this value click the grid cell and select another level from the list Units View the units used for quantity or concentration Retention Time 82 Quantitative Analysis User Guide View the retention time in minutes at the peak maximum Thermo Scientific A Quan Browser Reference The Quan Browser Window Table 9 Results grid parameters Sheet 2 of 2 Parameter Description Sample ID View the unique sample identification string Exclude Indicate whether to include or exclude the sample point from the calibration curve Include or exclude a point by clicking the grid cell select the check box to include the data or clear it to exclude the data Results Grid Shortcut Menu Table 10 Results grid shortcut commands Parameter Column Description View the list of possible columns that can be viewed in the Results grid view Placing a check next to an entry displays that column in the Results grid view of the Quan Browser window Rem
71. e letter abbreviation for the left edge type and a one letter abbreviation for the right edge type as follows Peak ID Left Edge Right Edge BB Baseline Baseline BT Baseline Tail B Tilt Baseline SS Stripe Stripe Flags Saturated View whether or not any of the scans in the peak were saturated The data system indicates one or more saturated scans were detected by displaying in the Saturated check box Calculated Amount View the amount of sample as calculated by the data system using the response ratio and Thermo Scientific the calibration curve Quantitative Analysis User Guide 105 A Quan Browser Reference Quan Browser Dialog Boxes Table 25 Flags page parameters Peak Information dialog box Sheet 3 of 3 Parameter Description Valley Detect View whether or not valley detection was turned on in the processing method The data system indicates that valley detection was activated if the Valley Detect check box is selected QC Failed View whether or not a sample failed a QC check If the calculated amount is greater than the specified percentage difference from the expected amount then a sample fails the QC test For example if the tolerance level is 10 and the expected amount is 100 calculated amounts less than 90 or greater than 110 fail The data system selects the check box to indicate that the sample type was QC and that it failed the QC test RT Ref OK View whether or not the Xcalibur data system found the re
72. e the settings on the Curve Isotope and Flags pages of the Calibration Settings dialog box In addition you can exclude one or more calibration points and one or more calibration levels from the from the calibration curve for each component Contents e Modifying the Calibration Settings e Setting Component Types in the Processing Setup Window e Setting Curve Parameters e Excluding Calibration Levels e Changing the Isotope Percentage Value e Including or Excluding Data Points from the Calibration Curve e Restoring an Excluded Data Point Modifying the Calibration Settings To modify the calibration for a target component 1 Open a sequence in the Quan Browser window 2 In the Components list select a target component The application automatically updates the Results grid and the Chromatogram and Companion views 3 To display only calibration standards in the Results grid click the Standards tab Thermo Scientific Quantitative Analysis User Guide 51 5 Working with Calibration Settings Modifying the Calibration Settings 4 If the Calibration Curve view is not displayed in the Companion view display it by doing one of the following e Choose View gt Set Companion View gt Show Calibration Curve e Right click the Chromatogram view and choose Set Companion View gt Show Calibration Curve from the shortcut menu 5 Inspect the calibration curve for the target component according to the criteria used in your
73. each sample in the Units box 9 To save the new settings and close the dialog box click OK These parameters are identical to those in the Target Compounds area on the Calibration page of Quan view in Processing Setup These are described in more detail in Calibration Settings Dialog Box on page 93 56 Quantitative Analysis User Guide Thermo Scientific Excluding Calibration Levels 5 Working with Calibration Settings Excluding Calibration Levels Use the Cal Exclusion List dialog box see Figure 27 on page 60 to exclude levels from the calibration For a description of the procedures used to generate this list see Understanding How Quan Browser Works on page 11 Excluding calibration levels is particularly useful when you cannot use the Include and Exclude commands because of overlapping points on the calibration curve If you are using a named calibration file levels might not be represented in the Results grid but will always be listed in the Cal Exclusion List dialog box To change the exclusion status of a level 1 Right click the Calibration Curve companion view and choose Exclusion List from the shortcut menu Figure 25 Cal Exclusion List dialog box Cal Exclusion List hydrocortisone Red Sox standard standardl standardJ Celtics Celtics standardL Expected 0 630 ng 2 029 1 250 ng 10 604 1 250 ng 2 920 2 500 ng 4 405 5 000 ng 0 560 5 000 ng 0 029 12 000 ng 2077 Yes Cancel Ap
74. ect file Select it and click Open The application opens the View Sample Types dialog box 3 To accept the All Sample Types default option click OK The application displays the Results grid Component List Chromatogram Plot and Spectrum Plot or Calibration Curve views 4 To select a component click a component in the Component List view The data system displays the chromatogram of the component in the Chromatogram Plot view 0 67 Not Found chromatogram peaks do not have blue baselines 0 67 Found chromatogram peaks have blue baselines 5 Examine the Chromatogram Plot view optional e To replot the chromatogram with a different X axis drag the cursor horizontally over the range that you want to expand The application rescales the axis and replots the data with the new X axis range e To replot the chromatogram with a different Y axis drag the cursor vertically over the range that you want to expand The application rescales the axis and replots the data with the new Y axis range e To cancel the replot and return to the full range of the X axis and the highest peak normalized to 100 0 on the Y axis click 4B 46 Quantitative Analysis User Guide Thermo Scientific Thermo Scientific 4 Working with Peaks Integrating Chromatogram Peaks Manually 6 To change the status of a peak from Found to Not Found optional right click the Chromatogram Plot view and choose Set Peak to Not Found Status from the
75. ected RT Component Expected RT Reference Actual RT Reference Expected This box is read only Quantitative Analysis User Guide 123 A Quan Browser Reference Quan Browser Dialog Boxes Table 36 Identification page parameters User Identification Settings dialog box Sheet 4 of 4 Parameter Description Detector Type Type View detector type options These are the detectors that you have configured using the Instrument Configuration dialog box Peak Detection This list contains three options from which you can select an Xcalibur peak detection Algorithm algorithm to recalculate the data using that algorithm Buttons Apply This button applies the current peak detection parameters to the selected component of the selected sample in the current sequence Apply to All This button applies the current peak detection parameters to all samples that are currently displayed in the sequence For example if Standards are displayed the current peak detection parameters are applied to only the Standards samples and not to any other samples If All samples are displayed the current peak detection parameters are applied to all samples in the current sequence If one or more Standard samples are changed the data system recalculates all quantitation parameters including peak areas and the calibration curve If one or more Standard samples are changed the data system recalculates all quantitation paramet
76. ed before the data system could determine the edge of the peak Unknown An unknown error occurred View the edge type on many reports using a one letter abbreviation for the left edge type and a one letter abbreviation for the right edge type as follows 104 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Table 25 Flags page parameters Peak Information dialog box Sheet 2 of 3 Parameter Description Peak ID Left Edge Right Edge BB Baseline Baseline BT Baseline Tail B Tilt Baseline SS Stripe Stripe Valid View whether or not the data system successfully detected the peak indicated by selecting the Valid check box Right Edge Type View how the data system detected the right baseline edge of the current peak The application displays one of the following peak baseline detection methods Edge Type Reported by Xcalibur Baseline B The edge of the peak is at baseline level Valley V The edge of the peak is in a peak valley Manual M The edge of the peak has been adjusted manually Stripe S The edge of the peak reached the Constrain Peak Height Percent specified in the method Tail T The edge of the peak reached the Constrain Peak Height Trailing Factor limit before the Height Percent Tilt An error occurred before the edge of the peak could be determined Unknown An unknown error occurred View the edge type on many reports using a on
77. ed by the BP and MR boxes In the case of a TIC no trace operator in use analog or digital traces this box is blank View specific flags stored in the processing method This is a read only field Set the Retention Time parameters e To change the minimum width that a peak is expected to have if valley detection is enabled type a new width in the Expected box e To change the time window or to enter a new time window type the number of seconds in the Window box e To change the current view width type the desired time in the View Width box Select a detector type from the Type list of detectors You configured this list using the Instrument Configuration dialog box To select an algorithm select a name in the Peak Detection Algorithm box and click OK The data system recalculates the current data using the specified algorithm The application changes the default parameters for peak detection to parameters specific to that algorithm 9 Click OK to save your settings Quantitative Analysis User Guide 39 4 Working with Peaks Setting User Integration Settings Setting Detection Values The application uses the parameters on the Detection page see Figure 18 to confirm the identity of the component within the retention time window defined by the Identification settings The options available on this page depend on whether the data is GC MS data or LC MS data To set detection values 1 From the User I
78. efault values for tolerance and precision on the Mass Options page of the Configuration dialog box Table 24 Masses dialog box parameters Parameter Mass tolerance Description Mass tolerance Specify the value for mass tolerance Type a value in the range of 0 1 to 50000 and select units to apply to the value The Xcalibur data system uses the tolerance value to create the limits of a range of masses Units Specify the units of measurement in which the data system processes your data Select mmu millimass units or ppm parts per million Mass precision Decimals Specify the number of decimal places mass precision that the data system uses to display mass values You can specify from 0 to 5 decimal places The number of decimal places applies to the mass data in a window Thermo Scientific Quantitative Analysis User Guide 101 A Quan Browser Reference Quan Browser Dialog Boxes Peak Information Dialog Box The Peak Information dialog box provides read only information about the currently displayed chromatogram peak or one of the peaks used by the spectrum search or ion ratio confirmation routines You can use this dialog box to quickly get detailed information on the found peak by selecting other components or other result files The current component name is displayed in the title bar for example Peak Information drugx If the current peak is for a Qualifier Ion the title bar also contai
79. ening as the sample travels through the column highly retained compounds produce wider chromatographic peaks So in general use a wider retention time window for late eluting compounds than for early eluting compounds Figure 2 shows the effect of retention time on peak width The chromatogram shown in Figure 2 contains four peaks The retention times and widths of these peaks are listed in the following table In this example hydrocortisone which elutes at 0 68 min has a peak width of 0 2 min whereas progesterone which elutes at 3 17 min has a peak width of 0 6 min 4 Quantitative Analysis User Guide Thermo Scientific Thermo Scientific 1 Overview of Quantitative Analysis Integrating and Identifying Chromatographic Peaks Figure 2 Integrated total ion current chromatogram TIC for steroidsO2 raw RT 0 00 3 79 SM 156 oO RT 0 68 NL 9 3 03E6 AA 14377780 100 TIC MS u 80 o c kad bad 5 99 RT 1 40 RT 3 17 3 2 40 AA 6556372 AA 11072975 E RT 1 99 a 20 AA 2447049 o 0 0 0 5 1 0 1 5 2 0 25 3 0 3 5 Time min Retention time Baseline peak width Compound min min hydrocortisone 0 68 0 2 deoxycorticosterone 1 40 0 3 methyltestosterone 1 99 0 4 progesterone 3 17 0 6 Because the Xcalibur data system might detect more than one chromatographic peak within the specified retention time window identify the target compound as either the highest peak in a chromatogram or the closest peak to the
80. er Value View or change the currently highlighted entry from the Value column in the event list The range of factors allowed for each value is specific to each event 134 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Table 40 Avalon Integration page parameters User Identification Settings dialog box Sheet 4 of 4 Parameter Page Buttons Description Add Add to add a time event value entry for a timed event in the event list When you click Add both the event list and the chromatogram display update automatically with the added specification in the currently selected chromatogram Delete Delete to remove a highlighted event from the event list You cannot delete initial values Change Thermo Scientific Change to update a highlighted time event value entry in the event list When you click Change both the event list and the chromatogram display update automatically with the revised specification in the currently selected chromatogram For initial events only the values and not the events can be changed Quantitative Analysis User Guide 135 A Quan Browser Reference Quan Browser Dialog Boxes Advanced Page User Identification Settings Dialog Box Use the Advanced page of the User Identification Settings dialog box to change the current advanced component detection criteria Use these additional criteria if the standard detection crite
81. ernal Standards nnna unuunu nanea 7 Chapter2 Quan Browser Overview 0 0 eeee eee eee eee eee e eens 11 Understanding How Quan Browser Works 0 0 0 0 cece eee eee 11 Calibration ReplicateS secure cerris eps eee Keene hanes her e 11 Named Calibration Filey ici caso cise ey cipe Boe be Cas oie Reo eee 12 Brackets and Groups for Sequences sau pcan cae ped deeded a ep eae ees 12 Unbracketed Sequence n n bad tee idadedacdeeede deaths badd 12 Open Bracket Seguenc tio iptv eed aida ea a e 14 Nonoverlapping Bracket Sequence n bdeadda ties deb ded eedes 14 Overlapping Bracket Sequence 0 0 cee ccc eee eee 14 Starting Quan Browser 1 eee e nee eee 15 Quan Browser Window Overview 0 0 0 0 cece cece nen eens 17 Component List 0 sca ck eee cote ea nae eee eed saw eae eeu eet 17 R s lts Grid ss con chawnatisen et dni EAE EEE EE dys 18 Chromatogram and Companion Views sic0ii4250csd0nsearaesase vows 19 Saving Changes Made in Quan Browser suuuuue ecer 21 Chapter3 Working with the Quan Browser Results Grid 0 a 25 Reviewing and Reworking the Results Grid 0 0 eee eee eee 25 Reviewing the Peak Status Column cascci ckietidokin keen ew eee enews 27 Making Changes in the Results Grid cade oowe aceon ceo vurs nto naes 28 Adding and Removing Samples 1 2 0 0 0 eee reene 28 Hiding or Displaying Columns 0 ccs cous ceed ees cons dhe cdeeeee nee 31 Changing the Sort Ofdetias ccc didd tea
82. ers User Identification Settings dialog box Sheet 1 of 2 Parameter Description Smoothing Points Type the number of points used in the moving average used to smooth the data The valid range is any odd value from 1 through 15 points The default value is 1 point This value is used by the ICIS peak detection algorithm Baseline Window Specify the number of scans over which to look for a local minima The valid range is 1 through 500 The default value is 40 scans This value is used by the ICIS peak detection algorithm Area Noise Factor Specify the noise level multiplier used to determine the peak edge after the location of the possible peak The valid multiplier range is 1 through 500 The default multiplier is 5 This value is used by the ICIS peak detection algorithm Peak Noise Factor Specify the noise level multiplier used to determine the potential peak signal threshold The valid multiplier range is 1 through 1000 The default multiplier is 10 This value is used by the ICIS peak detection algorithm Constrain Peak Width Limit the peak width of a component during peak integration of a chromatogram You can then set values that control when peak integration is turned on and off by specifying a peak height threshold and a tailing factor To constrain a peak width select the Constrain Peak Width check box The Peak Height box and the Tailing Factor box are activated Thermo Scientific Quantitative Analysis User Guide
83. ers including peak areas and the calibration curve 124 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Detection Page User Identification Settings Use the Detection page from the User Identification Settings dialog box to change the current peak detection criteria for component detection The parameters on the Detection page vary depending on whether you use a GC or LC whether the detection method is Spectrum Highest Peak or Nearest RT and the selected integration algorithm Table 37 Detection page parameters User Identification Settings dialog box Sheet 1 of 5 Parameter Spectrum Description This option is only available for GC MS data You can use a reference spectrum defined in the processing method for component identification The data system attempts to match the reference spectrum with a series of unknown spectra and calculates a score value for each comparison If you select the Spectrum option for GC MS data the following parameters appear e Spectrum Peak Detection Displayed only for GC MS data when you select the Spectrum option You must also have selected the MS detector type on the Identification page e Spectrum Peak Identification table Enter mass charge m z and intensity data for up to 50 spectrum peaks The data system uses this data to identify the active component in the Find algorithm It displays this table only when you select the
84. ew and change calibration settings reset the scaling of the view and copy the graph If you right click a calibration point the Calibration Point shortcut menu appears so that you can exclude include the point review calibration settings reset the scaling of the view or copy the graph Quan Browser Title Bar The title bar located in a horizontal band at the extreme top of the window contains the following e Application name e Current process e Current file name e Any optional parameters that are relevant For example if the active result file is ABC sld the data system might display Quan Browser Browser ABC sld Bracket 1 View All Quan Browser Menu Bar The Quan Browser menu bar provides access to these menus e File Menu e View Menu e Zoom Menu e Options Menu e GoTo Menu e Help Menu Thermo Scientific Quantitative Analysis User Guide 73 A Quan Browser Reference The Quan Browser Window File Menu Table 2 File menu commands Sheet 1 of 2 Command Open Description Open new data files The supported file types are sequence sld result rst and Quan Browser xqn files Save Create a new Xcalibur Quan Browser file with an xqn extension This file contains all the necessary information required to recreate the current browser session Save As Create a new Xcalibur Quan Browser file with a xqn extension This file contains all the necessary information required to recreate
85. expected retention time Use the Genesis and ICIS integration algorithms used for mass spectral data to rule out peaks below a specified signal to noise ratio 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 process 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 quantitation method is the lowest concentration of analyte in a sample that can be detected but not necessarily calculated as an exact value The lower and upper quantitation limits are the lowest and highest concentrations of analytes in a sample that can be measured with an acceptable level of accuracy and precision respectively In an analytical method the highest concentration calibration standard defines the upper quantitation limit The quantitation range is the range of concentration between the lower and upper quantitation limits including these limits that can be reliably quantified time after time with acceptable levels of accuracy and precision through the use of a concentration response relationship Quantitative Analysis User Guide 5 1 Overview of Quantitative Analysis Integrating and Identifying Chromatographic
86. ference The Quan Browser Window Right click a calibration point on the calibration curve in the Calibration Curve view to display the following shortcut menu hydrocortisone Y 0 557511 X R 2 0 9995 W Equal ve Exclude Calibration Settings Exclusion List Show Spectrum Plot Reset Scaling Area Ratio yo w A Oo Q N Copy Graph Table 14 Calibration Curve view shortcut commands Command Description Include Exclude Include or exclude data points on the calibration plot If the current data point was previously excluded the first menu item is Include instead of Exclude Select this command to include the data point and recalculate the calibration curve The data system updates the excluded entry in the Results grid to show that this point is now included and redraws the included data point on the calibration curve using a filled square If the data point is currently included in the calibration selecting the Exclude command forces the calibration curve to be recalculated without the selected data point The excluded entry in the Results grid view is updated to show that this point is excluded and the excluded data point is redrawn on the calibration curve using an unfilled square You can include or exclude samples that are shared between brackets Sample status is unique to the bracket that is excluding a shared sample in bracket 1 has no effect on its inclusion status in bracket 2 Cali
87. formation 1 In the Components list select the component of interest 2 Right click the Chromatogram view and choose Show Peak Info from the shortcut menu The Peak Information Dialog Box opens The Peak Information Dialog Box is read only If you select other components or samples the Xcalibur data system updates the dialog box with peak information for the displayed component chromatogram peak Figure 15 Info page Peak Information dialog box Peak Information hydrocortisone Info Flags More Flags Suitability Spectrum Left min a 60 Base Peak m z 203 2 Apex min a 68 Signal to Noise 707 56 Right min 0 85 Expected AT min 0 64 Height 00635 52 ISTD Response 6851 03 51 Area cts sec 1892942 73 Response Ratio 2 76 Baseline a 00 Calculated Amount 4 96 Close Help Quantitative Analysis User Guide 35 4 Working with Peaks Viewing Peak Information To check the validity of a chromatographic peak 1 Open the Peak Information dialog box for the component of interest as described above 2 Click the Suitability tab The Suitability page opens see Figure 16 Figure 16 Suitability page of the Peak Information dialog box Peak Information hydrocortisone Info Fags More Flags Suitability Spectrum Suitability Hags Symmetrical Not Tested Resolution Not Tested Peak Width Not Tested Tailing Not Tested a Column Overload Not Teste
88. h any of these sort options even if the corresponding column is not currently displayed For example you can sort by Sample Type even if you have selected the Sample Name check box in the Result List Column Hiding dialog box 3 Set any remaining column headings or file properties as the second and third sort criteria even if the column is currently hidden 4 To replace the default sorting criteria with your new selections click Save As Default 5 To sort the Results grid display click OK 32 Quantitative Analysis User Guide Thermo Scientific M Working with Peaks The Chromatogram view displays the chromatogram for the currently selected component from the currently selected result file Most of the commands for manipulating the Chromatogram view are available from a shortcut menu For shortcut menu details see Quan Browser Chromatogram Plot View on page 84 Contents e Viewing and Changing Peak Identification Detection and Integration e Setting the Chromatogram and Spectrum View Display Options e Viewing Peak Information e Setting User Integration Settings e Integrating Chromatogram Peaks Manually e Using the Spectrum Plot Companion View e Reviewing Spectrum Search Results for GC MS Data Viewing and Changing Peak Identification Detection and Integration After you acquire and process data files in the Sequence Setup view use the Quan Browser window to review the results of peak identification detection a
89. h uites Ak warees 57 Changing the Isotope Percentage Value cows idee eA tee eee ad 4 58 Including or Excluding Data Points from the Calibration Curve 59 Restoring an Excluded Data Point sa scyese vated actos don tir atata doin etacante aad 61 Viewing Results and Generating Reports 000ec eee eee ees 63 Reviewing the Results of the Quantitative Analysis 00 005 63 Generating Reports i yore ses tyke nade ete hae hee See os 66 Selecting Samples for Reports serie woe ata nin Wale ences Bnd 3 67 Printing REPOS bee Rei AGT ee Gey eae ein epee 68 Thermo Scientific Thermo Scientific Contents Appendix A Quan Browser Reference 0cee eee eeee eee eee eee ee eeeees n The Quan Browser Window 00 ccc ccc cece teen eee eens 72 Quan Browser Title Bar 0 0 6 cece ee een eee eees 73 Quan Browser Menu Bar 0 6 cece ence en eens 73 Pile Men si ssieres mirn oly a Ripe aed Mate eee NEEND EN ae ERS 74 View Menun ss psu min aches went at Rares Gea wiht hac ies eno a aie 75 Zoom Ment ies spn races CR wee RS ey elek Pew nae Alay 76 Options Ment cond i ose Gas a a ticles EA E E ian 76 GoTo M nu ie yee eh ala eco NS deco TG oren tae ehh e aa en TT Help Menu RG eon ao oP PN A RT ae ae 77 Quan Browser Toolbar 2 0 0 ccc ccc tte teen en eeas 78 Quan Browser Results Grid 20 00 ccc tent eas 81 Quan Browser Component List 0 0 0 ccc eee 84 Quan Browser Chromatogram
90. hat all scans from peak start to peak end are to be 138 Quantitative Analysis User Guide included in the area integration This value is used by the ICIS peak detection algorithm Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Flags Page User Identification Settings Dialog Box The Flags page of the User Identification Settings dialog box shows the current detection flagging thresholds in use for the selected compound These values are used in determining if the peak detection is within user specified limits They do not alter the way calculations are made An entered value of 0 0 forces the flag to be false Table 43 Flags page parameters User Identification Settings dialog box Parameter Description Area Threshold View or set a value for the current Area Threshold AT flag The data system sets the AT flag in the result file if the quantified peak has an area that is lower than the entered value Height Threshold View or set a value for the current Height Threshold HT flag The data system sets the HT flag in the result file if the quantified peak has a height that is lower than the entered value Thermo Scientific Quantitative Analysis User Guide 139 A Quan Browser Reference Quan Browser Dialog Boxes View Sample Types Dialog Box When opening Quan Browser the Xcalibur data system first checks to confirm that the file you select is valid After verifying that all the files exist and can be o
91. he Standards tab 6 Inspect the calibration curve in the Calibration Curve view Evaluate the calibration curve according to the criteria used in your laboratory e You can right click the Calibration Curve view to display commands in the shortcut menu for modifying the information in this view e You can right click a data point in the Calibration Curve view to display commands in the shortcut menu for modifying the data in this view 7 To select the first data file click the first row in the Results grid view Thermo Scientific Quantitative Analysis User Guide 63 6 Viewing Results and Generating Reports Reviewing the Results of the Quantitative Analysis 8 Check the entries in the Result grid view for peak detection and integration problems Ensure that the selected data file corresponds to the correct level and sample type You can right click the Results grid view to display commands in the shortcut menu for modifying the information in the Results grid view 9 Change the information in any of the following columns by clicking the appropriate grid cell e Inthe Sample Type column click a cell and select Standard QC Blank or Unknown from the list e In the Integration Type column click a cell and select Method Settings User Settings or Manual Integration from the list e In the Levels column click a cell and select another defined level from the list e In the Exclude column select or clear the Exclude check box
92. he acquisition data for this run Sample Type View the sample type that has been assigned to the sample Every sample must be designated one of these four basic types Standard QC Blank or Unknown You cannot change sample type of a sample that is shared between two brackets If you try to make this type of change the data system displays a warning message You cannot change the sample type of a sample shared between brackets Reverting back to original sample type Integration Type View the method the data system used to integrate the peak The options are Method User and Manual Select Method to apply the integration parameters defined during the processing setup phase Select User to override the integration parameters within Quan Browser Select Manual when the integration baseline was selected by dragging the baseline handles Area Or Height Internal Standard ISTD Area Height View the integrated area count seconds or height counts under the detected peak View the integrated area count seconds or height counts under the detected Internal Standard peak Area Ratio or Height Ratio View the area or height ratio between the selected peak and the Internal Standard peak Specified Amount Calculated Amount View the amount of the component at the Cal or QC level View the component amount as determined by the response ratio and calibration curve Difference View the percentag
93. he appearance of the Type page of the Calibration Settings dialog box changes depending on whether the selected component is a Standard or a Target Use the Type page for an Internal Standard component to change the Amount and Units settings that you specified on the Calibration page in the Quan view of the Processing Setup window Table 22 Internal Standard component parameters Parameter Component Type Description The data system recognizes two component types for component calibration purposes Target Compound components and Internal Standards ISTD components You can initially define select and characterize components of interest using the Calibration page in the Quan view of the Processing Setup window You can then revise these parameters using the Type page of the Calibration Settings dialog box available in the Quan Browser window Target Compound Specify that the selected component is a target compound This button is only active if you have defined at least one component as an internal standard and selected another component as a Target Compound component type Target Compound ISTD Specify that the selected component is an internal standard Internal Standard ISTD Amount View the amount of the selected component that is added to each sample to provide an internal standard Type amounts with up to three decimals of precision This box becomes active when you select the ISTD component type Units View
94. he calibration or quantitation is within user specified criteria They do not alter the way calculations are made An entered value of zero forces the flag to be false During entry of the values in the Quantitation Flags box the Xcalibur system checks to make sure that the relationships between the four fields are maintained If an entry in one field forces a change to occur in another field the Automatic Adjustment message box is displayed with the following message The last change made has forced an automatic change in one or more of the remaining edit boxes This is due to the fact that each value and the acceptable ranges for that value are linked Table 19 Flags page parameters Calibration Settings dialog box Parameter Calibration Flag Description R Squared Quantitation Flags Limit Of Detection View or change the current value of R squared The data system sets the R Squared RS flag in the result file if the computed coefficient of determination is lower than the entered R Squared value View or change the current value for the limit of detection The data system sets the Limit of Detection LOD flag in the result file if the peak concentration is less than the entered Limit of Detection Limit Of Quantitation View or change the current value for the limit of quantitation The data system sets the Limit of Quantitation LOQ flag in the result file if the peak concentration is less than the entered Limi
95. he current sequence 1 Click on the toolbar or choose View gt Reports dialog The Reports dialog box see Figure 28 duplicates the Reports view in Processing Setup When opened it displays the reports specified in the processing method associated with the active sequence The displayed parameters might change as you select different brackets Figure 28 Reports dialog box in the Quan Browser window wien Sample Reports 0 selected samples Enabled Stds QCs Unks Other Save As Report Template Name Yes Yes Yes Yes None Summary Reports Enabled Save As Report Template Name A none S O V Include Sample Reports E Include Summary Report Select Samples Print Reports Cancel Help 66 2 To include sample reports in any print run select the Include Sample Reports check box 3 To include summary reports in any print run select the Include Summary Reports check box 4 To open the Select Report Samples dialog box and choose samples in the sequence for report generation and printing click Select Samples For more information see Selecting Samples for Reports on page 67 5 To initiate report generation and printing as defined in the dialog box click Print Reports For more information see Printing Reports on page 68 Quantitative Analysis User Guide Thermo Scientific 6 Viewing Results and Generating Reports Generating Reports Selecting Samp
96. he initial value setting in the Time column These are the default integration events required by the Avalon integration algorithm You can change the Value of an initial entry integration event but you cannot delete it or change its time value Time This column contains either the term initial value or a value of time in minutes Event View descriptions of detection parameters for initial events and timed events You cannot change an event associated with an initial value Value View the values associated with initial events or timed events The range of factors allowed for each value is specific to each event Event List entry Time View or change the currently highlighted entry from the Time column in the event list Thermo Scientific Quantitative Analysis User Guide 133 A Quan Browser Reference Quan Browser Dialog Boxes Table 40 Avalon Integration page parameters User Identification Settings dialog box Sheet 3 of 4 Parameter Event Description View the currently highlighted entry in the Event column of the event list An event cannot be changed that is listed with an Initial Value in the Time column The Threshold and Bunch Factor parameters are the most important ones in controlling peak detection The data system provides the following events Start End Threshold Directly related to the RMS noise in the chromatogram this is Threshold the fundamental control used for peak detection Bunch Factor The Bunch
97. he spectrum at the right edge retention time of the current integration baseline choose Spectrum at Peak Right Edge e To change the companion view to display the calibration curve choose Show Calibration Curve e To display the full spectrum in a normalized window choose Reset Scaling 48 Quantitative Analysis User Guide Thermo Scientific 1M kod 4 Working with Peaks Reviewing Spectrum Search Results for GC MS Data To rescale the plot Use the cursor to select an area to magnify or use the Zoom menu commands and buttons on the toolbar Reviewing Spectrum Search Results for GC MS Data The Xcalibur data system only performs ion spectrum searches on GC MS data bd L Thermo Scientific To review the results of a spectrum search in Quan Browser From the Quan Browser window choose File gt Open The Open dialog box opens From the Files of Type list select either sld rst or xqn Browse to find the file you want to review Select it and click Open The View Sample Types dialog box opens Select the Show All sample types option and click OK To select a component to review click a component in the Component list view that has had a spectrum search as specified in the Processing Setup method The application displays the chromatogram in the Chromatogram Plot view To view the plot right click the Chromatogram Plot view to display the shortcut menu Depending upon the spectrum search results the d
98. hic Peaks Thermo Scientific Quantitative Analysis User Guide 1 1 Overview of Quantitative Analysis Acquiring and Quantitatively Processing Data Acquiring and Quantitatively Processing Data Quantitative analysis is the process of measuring the amount of a particular component in a sample With the Xcalibur data system quantitative analysis usually involves the following steps Note The order of some of these steps is not rigid For example you can acquire and process a set of data files using a sequence that contains both an instrument method and a processing method and you can print reports without previewing them first For more information refer to the Xcalibur Data Acquisition and Processing User Guides 1 Create an instrument method The Xcalibur data system uses an instrument method to store a specific set of parameters used to operate the autosampler LC pump or MS pump gas chromatograph mass spectrometer PDA detector and so on For more information about creating an instrument method refer to your hardware documentation 2 Create an acquisition sequence An acquisition sequence identifies the position of the samples in an autosampler tray if appropriate the instrument method used to control the HPLC GC MS or LC MS instrument and the directory and file names for the acquired data files For more information about creating an acquisition sequence refer to the Xcalibur Data Acquisition and Processing Use
99. ht click the Spectrum Plot view and select Spectrum At Peak Left Edge or Spectrum At Right Edge from the shortcut menu Table 12 describes the shortcut menu commands For more information about working with the Spectrum Plot view see Using the Spectrum Plot Companion View on page 48 Table 12 Spectrum Plot view shortcut commands Command Spectrum At Peak Apex Description View the spectrum at the apex retention time of the displayed chromatogram peak Spectrum At Peak Left Edge View the spectrum of the left edge of the displayed chromatogram peak The left edge is defined at the retention time corresponding to the left baseline box handle Spectrum At Peak Right Edge View the spectrum of the right edge of the displayed chromatogram peak The right edge is defined at the retention time corresponding to the right baseline box handle Display Options Select the style color labels axis and normalization options for your spectrum from the Display Options dialog box This menu command is not active if no spectrum data is displayed in the Spectrum Plot view Show Calibration Curve Reset Scaling View the Calibration Curve view in the lower right corner of the Quan Browser window Reset the plot scale to include the full peak in a normalized window 86 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference The Quan Browser Window Quan Browser Calibration Curve View
100. in a cell to exclude or include the sample in the bracket calibration Selecting excludes the data and is indicated in the grid by Yes When a sample is shared between two brackets you cannot change its sample type The data system notifies you when a sample is part of two overlapping brackets if you attempt to change its Integration Type Level or Exclude state 10 Inspect the component peak in the Chromatogram Plot view e Make sure that the data system found the peak The data system shades all found peaks gray and marks the starting and ending points with square integration markers e Make sure that the data system integrated the peak properly The shaded area should accurately represent the contribution of the component to the chromatogram For more information about identifying detecting and integrating peaks see Chapter 4 Working with Peaks If necessary perform steps 11 and 12 11 To modify the peak detection and integration settings optional do the following a Right click the Chromatogram Plot view to display commands in the shortcut menu for modifying the information in this view b Choose User Peak Detection Settings to display the User Identification Settings dialog box For more information see Setting User Integration Settings on page 37 64 Quantitative Analysis User Guide Thermo Scientific Thermo Scientific 15 6 Viewing Results and Generating Reports Reviewing the Results of the Quant
101. isplayed in the Calibration Curve view 92 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Calibration Settings Dialog Box The Xcalibur data system displays particular pages in the Calibration Settings dialog box based on whether the component is an Internal Standard or a Target e Available pages if the component is an internal standard Type Page Calibration Settings Dialog Box e Available pages if the component is a target Curve Page Calibration Flags Page Calibration Isotope Page Calibration Settings Dialog Box Settings Dialog Box Settings Dialog Box Levels Page Calibration Type Page Calibration Settings Dialog Box Settings Dialog Box Curve Page Calibration Settings Dialog Box Use the Curve page to modify component type settings that you specified on the Calibration page in the Quan view of the Processing Setup window To test the results of the new setting click Apply or OK Table 18 Curve page parameters Calibration Settings dialog box Sheet 1 of 2 Parameter Description Calibration Curve View the selected calibration curve type Specify how to use the origin in your calibration curve You can choose to ignore the origin Origin as a data point force the curve to pass through it or include it as a single point on the calibration curve Ignore Exclude the origin as a valid point in your calibration curve If you select
102. itative Analysis c Doone or more of the following e To modify the peak detection settings click the Detection tab e Ifyou have problems with noise in the peak or peak tailing click the Integration tab to modify the settings e Ifbaseline noise is interfering with peak identification or integration click the Advanced tab to modify the settings To manually change the starting and ending points and baseline of the peak drag the square integration markers to the desired location To review the next data file click the next row in the Results grid view Go to step 8 To review and rework the QC results do the following a Click the QCs tab b Perform steps 7 through 13 for the QCs Evaluate the QCs according to the criteria used in your laboratory To review and rework the Blank results do the following a Click the Blanks tab b Perform steps 7 through 13 for the blanks Evaluate the blanks according to the criteria used in your laboratory To review and rework the Unknown results do the following a Click the Unknowns tab b Perform steps 7 through 13 for the Unknowns Evaluate the Unknowns according to the criteria used in your laboratory For each remaining component select the component in the Components list and then perform steps 8 through 15 Quantitative Analysis User Guide 65 6 Viewing Results and Generating Reports Generating Reports Generating Reports To generate reports for t
103. its of counts for the current component peak read only Response Ratio View the ratio of the peak sample peak area or height to the internal standard area or height read only Calculated Amount View the amount of sample calculated by the Xcalibur data system using the response ratio Thermo Scientific and the calibration curve read only Quantitative Analysis User Guide 107 A Quan Browser Reference Quan Browser Dialog Boxes Info More Info Page Peak Information Dialog Box The Info More Info page of the Peak Information dialog box is displayed if you have conducted a spectrum search e Ifthe main component peak was detected using the spectrum search then this page has the More Info tab and displays only the Spectrum box In this case the pages displayed are Info More Info Flags More Flags Suitability and Spectrum e If peaks other than the main component were detected using the spectrum search then this page has the Info tab and displays both the Spectrum box and the Peak Info box In this case the pages displayed are Info Chro and Spectrum Use this page to review the following peak properties for the current search component Table 27 Info More Info page parameters Peak Information dialog box Parameter Spectrum Results Description View the calculated Forward Reverse and Match results for the found peak read only This box appears on the More Info page for Candidate 1 of a spectrum search
104. l standard compound To determine this ratio experimentally analyze the ISTD reagent using the method to be used for quantitation of the target compound Use the respective peak areas or heights to determine the ratio of impurity to pure compound The valid range is 0 00 to 100 00 percent To change the impurity ratio type a new value in the Contribution of ISTD to Target Compound box The data system uses this ratio as the value x in the following impurity correction expressions ISTD corr ISTD obs y TM obs 1 yx TM corr TM obs x ISTD obs 1 yx Where ISTD corr is the corrected amount of internal standard ISTD obs is the apparent amount of ISTD as measured by the application at the retention time for ISTD This peak consists of ISTD corr TM impurity TM corr is the corrected amount of the target molecule TM obs is the apparent amount of TM as measured by the application at the retention time for TM This amount consists of TM corr ISTD impurity See Contribution of Target Compound to Internal Standard box for a complete description of the variable y 96 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Table 20 Isotope page parameters Calibration Settings dialog box Sheet 2 of 2 Parameter Contribution of Description Target Compound To ISTD Thermo Scientific View the ratio TM impurity TM pure
105. le Type and Level lists and you can exclude or include calibration points To change the text in the Sample Name column for a sample delete the sample from the grid and then add it back to the grid as described in the next topic Adding and Removing Samples To change the integration select Method Settings or User Integration in the Integration Type column You can select Manual Integration after you manually integrate a peak as described in Integrating Chromatogram Peaks Manually on page 46 When you select Method Settings Quan Browser integrates the component peak by using the settings in the current processing method When you select User Integration Quan Browser integrates the component peak by using the settings in the User Identification Settings dialog box For information about using the User Identification Settings dialog box see Setting User Integration Settings on page 37 Adding and Removing Samples 28 To add samples to the Results Grid 1 To minimize data entry select a sample in the Results grid that it similar to the sample or samples that you are adding 2 Right click the Results grid to display the shortcut menu and choose Add Sample The Open Rawfile dialog box opens Quantitative Analysis User Guide Thermo Scientific 3 Working with the Quan Browser Results Grid Adding and Removing Samples 3 Browse to the appropriate folder and select the raw file or raw files to add to the sequence
106. lect to include or exclude data points in the calibration curve e To include an excluded point click Yes in the Exclude column for the data point The application changes the Excluded Yes to Excluded Blank e To exclude an included point click the Exclude column for the data point The data system changes Excluded Blank to Excluded Yes 6 To replot the calibration curve and recalculate results click Apply The application plots included data points as filled squares and excluded data points as outlined squares replots the calibration curve using only included points and recalculates the values for Calculated Amount Diff and RSD in the Results grid 7 Repeat steps 5 and 6 until you obtain the desired results 8 To close the Cal Exclusion List click OK Note When you close Quan Browser your selections and recalculations are erased Save your selections and recalculations by choosing File gt Save As to create a Quan Browser file xqn 60 Quantitative Analysis User Guide Thermo Scientific 5 Working with Calibration Settings Restoring an Excluded Data Point Restoring an Excluded Data Point Thermo Scientific To restore a data point that you have previously excluded right click the data point and choose Include from the shortcut menu The Xcalibur data system does the following e Incorporates the data point into the calibration and recalculates the curve e Updates the corresponding Peak Statu
107. les for Reports After clicking Select Samples in the Reports dialog box see Figure 28 use the Select Report Samples dialog box Figure 29 to choose the samples to be processed during report generation Figure 29 Select Report Samples dialog box Select Report Samples Sample Choices Selected Samples steroids02 Standard steroids03 Standard steroids04 Standard steroids05 Standard steroids13 Standard steroids14 Standard steroids15 Standard steroids16 Standard sampled Unknown sampleB Unknown Add All gt gt sampleC Unknown sampleD Unknown sampleE Unknown lt lt Remove All OK Cancel Help To include a sample in report processing 1 Select the sample name in the Sample Choices list 2 Click Add to add the sample in the Sample Choices list to the Selected Samples list You can add all of the samples by clicking Add All Select multiple files using the SHIFT and CTRL keys as follows e To select a range of samples hold the SHIFT key down e To select multiple samples hold the CTRL key down To exclude a sample from report processing 1 Select the sample name in the Selected Samples list 2 To remove the sample from the Selected Samples list click Remove You can remove all of the samples from the Selected Samples list by clicking Remove All Thermo Scientific Quantitative Analysis User Guide 67 6 Viewing Results and Generating Reports Generating Reports Printing Reports N DB ww 10 1
108. llowing e From the Quan Browser menu choose View gt Set Companion View gt Show Spectrum Plot e Right click the Companion view and choose Show Spectrum Plot from the shortcut menu b View spectra across the chromatogram Pin the Spectrum Plot Companion view Click points of interest in the chromatogram to view the corresponding spectrum See Using the Spectrum Plot Companion View for more information 6 To do a detailed qualitative analysis of the chromatogram export the result file to Qual Browser Right click the Results grid and choose Send to Qual Browser from the shortcut menu For more information about using Qual Browser refer to the Xcalibur Qualitative Analysis User Guide Setting the Chromatogram and Spectrum View Display Options With the exception of the Composition options for the Spectrum view the display options for the Chromatogram and Spectrum views in Quan Browser are the same as those in Qual Browser For information about changing the display options for the chromatogram and spectrum views refer to the Xcalibur Qualitative Analysis User Guide 34 Quantitative Analysis User Guide Thermo Scientific 4 Working with Peaks Viewing Peak Information Viewing Peak Information Thermo Scientific Quan Browser displays information about the currently displayed component peak qualifier ion or spectrum candidate in the Peak Information dialog box The title bar contains the component name To view peak in
109. me in the Calibration File box Although in theory a different calibration file name for every sample is possible in practice only one name per sequence is common Named calibration files are not available with bracketed sequences Brackets and Groups for Sequences This section describes different bracket types and when to use them to get a specific result e Unbracketed Sequence e Open Bracket Sequence e Nonoverlapping Bracket Sequence e Overlapping Bracket Sequence Note Use the New Sequence Template dialog box in the Sequence Setup view of the Home Page window to set up sequence bracketing for the None Non Overlapped and Overlapped bracket types Use either the New Sequence Template dialog box or the sequence table in the Sequence Setup view to set up bracketing for the Open bracket type Unbracketed Sequence The Xcalibur data system processes an unbracketed sequence None bracket type using a procedure known as the continuing calibration method Each time the application processes an unbracketed sequence it creates or updates the calibration files named in the sequence Select this process and avoid using Std Clear Standard Clear to add replicate data incrementally to a calibration file without discarding the existing replicate data The Quan Browser application breaks down unbracketed sequences into logical groups that are somewhat analogous to brackets It does this by first ordering the samples chronologically with re
110. mples appear in the Results grid according to the sort order specified in the Quantitation Results Sorting Order dialog box When you add a standard Quan Browser recalculates the calibration curve for the bracket and the calculated amounts for the samples within the bracket For information about changing the sort order see Changing the Sort Order on page 32 and Quantitation Results Sorting Order Dialog Box on page 112 8 When you finish you edits save your changes as described in step 8 of Reviewing and Reworking the Results Grid on page 25 30 Quantitative Analysis User Guide Thermo Scientific 3 Working with the Quan Browser Results Grid Hiding or Displaying Columns To remove samples from the sequence Select the row or rows in the sequence to delete Right click the sequence to display the shortcut menu and choose Delete Selected Samples When you finish your edits save your changes as described in step 8 of Reviewing and Reworking the Results Grid on page 25 Hiding or Displaying Columns The Quan Browser Results grid contains many columns Choose to display some or all of these columns using the Result List Column Hiding dialog box Figure 13 Result List Column Hiding dialog box Result List Column Hiding Thermo Scientific Selected Columns File Name Percent Difference Sample Type Percent RSD Sample Name Peak Status Integration Type g Area Height ISTD Area Height Area
111. n filter command string into the box using the scan filter format For example the following scan filter c full ms 26 81 251 finds all scans in a raw file that have the following properties centroid data Scan Mode Full Scan Power MS Product Ion Mass Range m z 26 81 to 251 00 The data system displays this box when you select a Base Peak trace for an MS detector type The box displays the range within which the application is to search for the highest peak If you type a single m z value in this box that defines the base peak To change the base peak mass range type the value in the box A mass range from m z A to m z B is entered in the format A B The data system displays this box when you select a Base Peak Mass Range trace combination for an MS detector type The box displays the mass range for the second Mass Range trace type To change the range or to add a new range type the range in the box The format is Low Mass High Mass For example for the range m z 123 through 456 type 123 456 Mass This box displays the masses stored in the processing method This display area changes to accommodate the type of data required When a single mass range is required there is a single edit box displaying the current value If two mass ranges are required such as the case of a trace defined as a Mass Range Mass Range or Base Peak Mass Range this box is replaced by two boxes in the case of Base Peak Mass Ra
112. n opens with the report displayed behind the Quan Browser window and Quan Browser automatically saves the spreadsheet to the current working directory To save the spreadsheet with a name other than the default use the Save As command Quantitative Analysis User Guide 23 M Working with the Quan Browser Results Grid This chapter describes how to modify the Quan Browser sequence by changing the sample type for each row changing the level for standards and QC samples adding and deleting rows and changing the sort order This chapter also describes how to use the Add Sample dialog box to change several parameter settings in a result file including the Sample ID Sample Name ISTD Corr Amt and Dilution Factor For information about excluding standards from the calibration curve for a component see Including or Excluding Data Points from the Calibration Curve on page 59 Contents e Reviewing and Reworking the Results Grid e Adding and Removing Samples e Hiding or Displaying Columns e Changing the Sort Order Reviewing and Reworking the Results Grid To review and edit a sequence 1 To open a sequence in the Quan Browser window do the following a Choose File gt Open The Open dialog box appears b In the File of Type list select one of the following file types e Sequence List Files sld e Result Files rst e Quan Browser Files xqn Quantitative Analysis User Guide 25 3 Working with the Quan Brow
113. n peak by comparing the relative responses of qualifier ion and quantitation peaks with predetermined values Window Use this column in the Qualifier Ion table to specify the Target Ratio tolerance for a qualifier ion Use other rows of the table to enter data for five qualifier ions The data system uses this data to confirm the identity of a quantitation peak by comparing the relative responses of qualifier ion and quantitation peaks with the specified values Thermo Scientific Quantitative Analysis User Guide 127 A Quan Browser Reference Quan Browser Dialog Boxes Table 37 Detection page parameters User Identification Settings dialog box Sheet 4 of 5 Parameter Description Window Select Relative or Absolute e Relative Specify that the target ratio tolerance values in the Window column of the qualifier ion table are relative values For example if the target ratio is 50 and the Window parameter is 20 the expected target ion ratio range is 40 to 60 with the Absolute option this would be 30 to 70 If the ion ratio is outside this range the ion ratio confirmation test has failed and the data system sets the IRC Flag to false If the qualifier ion peak quantitation peak ratio is within range the ion ratio confirmation test passes and the application sets the IRC Flag to true The response of all specified qualifier ions must be within the respective ratio ranges for IRC to succeed In assessing a target ion r
114. n the chromatogram for component identification Nearest RT This option is available for both LC MS and GC MS data Use the peak with the nearest retention time in the chromatogram for component identification lon Ratio Confirmation GC MS data If you are working with GC MS data and you select the Highest Peak or Nearest RT options the Ion Ratio Confirmation area appears You must also have selected the MS detector type on the Identification page Enable Indicates whether or not Ion Ratio Confirmation is enabled Ion Ratio Using Area or Height View the currently selected peak quantitation method area or height The data system uses the same method to calculate qualifier ion peak response and then target ratio You can change this parameter by selecting the Area or Height options in the Response box on the Calibration page 126 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Table 37 Detection page parameters User Identification Settings dialog box Sheet 3 of 5 Parameter Description Qualifier Ion Table Specify up to five qualifier ions in this box to confirm the detection of a target analyte You can also set the coelution window and select a method for calculating the target ion ratio window and tolerance Use this table to enter mass charge 7 z and target ratio tolerances Window data for up to five qualifier ions e Ifyou are using Area
115. nd integration To review and rework peak identification detection and integration in Quan Browser 1 In Quan Browser open a file result file sequence file or Xcalibur Quan file select a component in the Component list and select a sample in the Results grid 2 Right click the Chromatogram view and choose Show Peak Info from the shortcut menu The Peak Information Dialog Box opens Thermo Scientific Quantitative Analysis User Guide 33 4 Working with Peaks Setting the Chromatogram and Spectrum View Display Options 3 Review the chromatogram peak data in these areas e The properties of the detected peak on the Info page e The integration information and flags on the Flags page e The System Suitability test results on the Suitability page e The spectrum for the peak apex scan on the Spectrum page 4 Adjust the peak detection and integration in the Chromatogram view e Change detection or integration parameters See Setting User Integration Settings on page 37 e Manually integrate peaks See Integrating Chromatogram Peaks Manually on page 46 e Change chromatogram peak labeling To change the labels right click the Chromatogram view and choose Display Options from the shortcut menu In the Display Options dialog box click the Labels tab On the Labels page select the labels to display 5 To view spectra across the peak do the following a Display the Spectrum Plot companion view by doing one of the fo
116. ndard Method Settings 6 drugx_08 Standard Method Settings 9 drugx_09 Standard Method Settings 10 drugx_10 Standard Method Settings 11 drugx_11 Standard Method Settings 12 drugx_12 Standard Method Settings 13 drugx_13 Standard Method Settings qA A A Standards A OCs oo oo SyS In the Options menu if you choose to view to View Stds and QCs the Results grid view displays three tabs All Stds and QCs The All page displays all Stds and QCs the Stds page displays only standards and the QCs page displays only QCs In the Options menu if the viewing preference is set to View All the Results grid view displays five tabs All Stds QCs Blanks Unknowns The All page in this case displays all Stds QCs Blanks and Unknowns The Stds page displays only standards the QCs page displays only QCs the Blanks page displays only blanks and the Unknowns page displays only unknowns Depending on the current settings of the Result List Column Hiding dialog box the Results grid view can display different parameters for each sample row These topics describe the columns in the Results grid view and the shortcut menu e Results Grid Columns e Results Grid Shortcut Menu Thermo Scientific Quantitative Analysis User Guide 81 A Quan Browser Reference The Quan Browser Window Results Grid Columns Table 9 Results grid parameters Sheet 1 of 2 Parameter File Name Description View the raw file that contains t
117. nge this box is replaced by the BP and MR boxes In the case of a TIC no trace operator in use Analog or Digital traces this box is blank Keys This box displays specific flags stored in the processing method This is a read only field used for reference 122 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Table 36 Identification page parameters User Identification Settings dialog box Sheet 3 of 4 Parameter Retention Time Description Retention Time The settings in this area define the expected retention time in minutes of the component 8 p P peak and the error window in seconds for the retention time Expected Window This box displays the expected peak width parameter in seconds This controls the minimum width that a peak is expected to have if valley detection is enabled With valley detection enabled any valley points nearer than the expected width 2 to the top of the peak are ignored If a valley point is found outside the expected peak width the data system terminates the peak at that point It always terminates a peak when the signal reaches the baseline independent of the value set for the expected peak width The valid range is 0 0 to 999 0 seconds To change the current value type a new width in the Expected box This box displays the allowable retention time window for the elution of the selected component The valid range is 1 0 t
118. non ISTD components in the sample The concentration or amount of an ISTD in any standard or unknown sample typically remains constant Because quantitative mass spectrometric analysis usually involves multiple steps the total error in the analysis results from the accumulation of errors at each step In general sample handling errors account for a larger fraction of the total error than detector errors do 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 Lack of injection reproducibility e Changes in analyte solution volume e Matrix and coeluter interference both suppression and enhancement e System instability e Variations in the source conditions Quantitative Analysis User Guide 7 Quantitative Analysis User Guide 1 Overview of Quantitative Analysis Integrating and Identifying Chromatographic Peaks For maximum precision add the ISTD component 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 and since the ISTD is known and quantifiable the internal standard quantitation approach corrects for injection and other sample handling errors The ISTD must behave chemically in an identical or similar manner
119. ns the text Qual Ion Mass xxx x where the xxx x represents the mass of the qualifier ion If the peak is for a spectrum candidate the title bar also contains the text Spectrum Candidate The pages displayed in the Peak Information dialog box depend on the type of peak that is currently displayed as follows e No Peak e Standard Peak e Qualifier Ion Peak e Spectrum Candidate Peak First Candidate e Spectrum Candidate Peak Second and Third Candidates No Peak If no peak has been found for the component the Xcalibur data system displays only the No Peak page with the following message No Peak Found Cannot show Peak Info No Peak Page Peak Information Dialog Box 102 Quantitative Analysis User Guide Thermo Scientific Standard Peak A Quan Browser Reference Quan Browser Dialog Boxes This series of pages is only available if the inlet is a liquid chromatograph Note When a liquid chromatograph is used for the inlet the Ion Ratio Confirmation option is not available Info Page Peak Information Dialog Box Suitability Page Peak Information Dialog Box Qualifier lon Peak Flags Page Peak Information Dialog Box Spectrum Page Peak Information Dialog Box More Flags Page Peak Information Dialog Box This series of pages is only available if the inlet is a gas chromatograph Note Along with the five pages used for a standard peak the More Info page and Chro page are availa
120. ntification Settings dialog box click the Flags tab Figure 21 Flags page User Identification Settings dialog box User Identification Settings Identification Detection Genesis Integration Genesis Advanced Flags Area Threshold Height Threshold o o Cancel Apply Apply Toal Help 2 To test the validity of detected peaks check values on the Flags page 3 Type a value in the current Area Threshold AT box The data system sets the AT flag in the result file if the quantified peak has an area that is lower than the entered value uN Type a value in the current Height Threshold HT box The application sets the HT flag in the result file if the quantified peak has a height that is lower than the entered value 5 To save the settings in a Quan Browser file xqn choose File gt Save or File gt Save As ion To export the user settings as a full processing method choose File gt Export Method 7 Click OK to save your changes Thermo Scientific Quantitative Analysis User Guide 45 4 Working with Peaks Integrating Chromatogram Peaks Manually Integrating Chromatogram Peaks Manually To modify and test component peak integration criteria 1 To open the sequence file that contains the calibration standards from the Quan Browser window choose File gt Open The application displays the Open dialog box 2 In the Files of Type list select Sequence list files Browse to find the corr
121. o 999 0 seconds To change the time window or to enter a new time window type the number of seconds in the retention time Window box Use as RT Reference This check box indicates whether or not the actual retention time RT of the active component as displayed in the Name combo box in the same view was used to adjust the expected retention time of another component This check box is read only View Width Adjust Using This box displays the current view width in minutes The valid range is dependent upon the configured hardware To change the view width type the desired time in the View Width text box This check box indicates whether or not the expected retention time RT of the active component as displayed in the Name box in the same view is to be adjusted using the actual retention time of the RT Reference such as an internal standard The data system displays the RT Reference in the Adjust Using box to the right of this check box This check box is read only Adjust Using box Thermo Scientific This box displays the retention reference component that Xcalibur uses to adjust the expected retention time of the active component as displayed in the Name box in the same view The data system uses the actual retention time of the RT Reference component to correct the retention time of the active component The application provides the following correction to the expected retention time Adjusted RT Component Exp
122. o correct data in these situations e An impurity in the internal standard compound that elutes at the same time as the target compound e An impurity in the target compound that elutes at the same time as the internal standard These parameters are identical to those in the Correction For Isotope Contribution dialog box accessed from the Calibration page of Quan view in Processing Setup To set Isotope parameters 1 In the Quan Browser window show a calibration curve in the Companion view 2 Right click the Companion view and choose Calibration Settings from the shortcut menu Click the Isotope tab Figure 26 Isotope page Calibration Settings dialog box Calibration Settings Type Curve Levels Isotope Flags Contribution of ISTD to Target Compound Target Compound to ISTD 0 0 58 Quantitative Analysis User Guide Thermo Scientific 5 Working with Calibration Settings Including or Excluding Data Points from the Calibration Curve 3 Choose from these options e Ifyou have an impurity in your internal standard that elutes at the same time as the target molecule use the ISTD to Target Compound box to type the ratio ISTD impuritylISTD pure To determine this ratio experimentally analyze the ISTD reagent using the method to be used for quantitation of the target compound Use the respective peak areas or heights to determine the ratio of impurity peak at retention time of TM to pure compound pe
123. ompanion view you can choose either the Spectrum Plot view or the Calibration Curve view Figure 9 shows the Calibration Curve view and shortcut menu Figure 9 Calibration Curve view and shortcut menu hydrocortisone Y 0 557511 X R 2 0 9995 W Equal 7 6 n Calibration Settings 5 Save Calibration File 4 ee Exclusion List 3 Show Spectrum Plot 2 Reset Scaling 1 Copy Graph 0 0 2 4 6 8 10 12 ng 20 Quantitative Analysis User Guide Thermo Scientific 2 Quan Browser Overview Saving Changes Made in Quan Browser Saving Changes Made in Quan Browser In the Quan Browser window you can do the following Modify result files rst Modify processing methods pmd e Create Xcalibur Quan files xqn e Export the quantitation results in the Results grid to an Excel spreadsheet e Set up new sample sets in the Results grid by adding or deleting samples however you cannot export the modified sample set as a new sequence file sld To continue working with a modified sample set you must save your changes to an Xcalibur Quan file xqn e Evaluate manual integration settings for component peaks however you cannot save the results of manual integration to a result file To continue working with manually integrated peaks you must save your changes to an Xcalibur Quan file xqn For information about changing some of the information associated with the result file such as the sample type sample name
124. or calibration level see Making Changes in the Results Grid on page 28 For information about changing the peak identification detection and integration settings see Viewing and Changing Peak Identification Detection and Integration on page 33 For information about changing the calibration settings see Modifying the Calibration Settings on page 51 To save changes to the result files After you make changes to result files save the changes by choosing File gt Save All To save the changes to an Xcalibur Quan file 1 Choose File gt Save The Save As dialog box opens 2 Select an appropriate directory in the Save In box and type an appropriate name in the File Name box 3 To save the file and close the Save As box click Save Thermo Scientific Quantitative Analysis User Guide 21 2 Quan Browser Overview Saving Changes Made in Quan Browser To save the changes to an Xcalibur Quan file and set up an audit trail 1 After making changes save a file with the changes by choosing File gt Save As The File Summary Information dialog box opens Figure 10 File Summary Information dialog box File Summary Information Header treated ast saved by umber of saves Sunday October 10 2004 8 41 43 PM Cook Not changed Comment 2 Enter a comment clearly identifying the changes you made and click OK The Save As dialog box opens Figure 11 Save As dialog box Save in
125. ort orders to display the Results grid view until you use the Quantitation Results Setting Order dialog box to change your column sorting preferences Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Reports Dialog Box Use the Reports dialog box to generate reports on each sample row within the current bracket The Reports dialog box opens with a selection of Report templates and data files preloaded These are obtained from the processing method that was previously defined and loaded along with the result file This means that the values loaded might change as various brackets are selected Note Prior to using the Reports dialog box you must create a reports template For additional information on how to create a report template see the XReport User Guide Table 32 Reports dialog box parameters Sheet 1 of 4 Parameter Sample Reports Description Enabled Specify whether or not the reports marked with Yes in their row are processed using the template that appears in the Report Template Name box For example if Yes appears in the QCs and Unknowns boxes and the Enable box in the row displays Yes then the data system prints these sample reports If the Enabled box in the row is clear then the application does not print any sample reports When you click an Enabled box a check box control appears If you select this check box and you click another cell the application displays the word Yes to
126. oving the check removes the column from the Results grid view Delete Selected Samples Remove the currently selected samples from the Results grid view If you delete standard samples the data system recalibrates the data If you delete either standard samples stds or quality control samples QCs the application recalibrates the Relative Standard Deviation Add Sample Select a new file from a browse dialog box The data system adds it to the Results grid view and sorts it according to the sort order If you add standard samples the application recalibrates the data If you add either standard samples stds or quality control samples QCs the Xcalibur system recalibrates the Relative Standard Deviation Copy Row Duplicate the currently selected row and add it to the next row of the Results grid view If the row added is a standard sample std then a recalibration takes place If it is either a standard sample std or a quality control sample QC then the data system recalculates the Relative Standard Deviation Set Sorting Order Set the sort order for the samples in the Results grid view Send To Qual Browser Thermo Scientific Open the result file for the sample that you selected in Qual Browser Quantitative Analysis User Guide 83 A Quan Browser Reference The Quan Browser Window Quan Browser Component List The components list on the right side of the window lists the components in the p
127. owing peak properties for the current component Table 28 More Flags page parameters Peak Information dialog box Parameter Detection Thresholds Description Area Set a specified area tolerance value to compare with the peak area and determine if the peak area is outside or within the tolerance Height Cal and Quan Thresholds R Squared Set a specified height tolerance value to compare with the peak height and determine if the peak height is outside or within the tolerance Set a specified coefficient tolerance value to compare with the coefficient of determination and determine if the coefficient of determination is outside or within the specified tolerance Detection Limit Linearity Limit Set a specified detection threshold tolerance value to compare with the concentration of the quantified peak and determine if the detection threshold is outside or within the specified tolerance Set a specified linearity threshold tolerance value to compare with the linearity threshold for the concentration of the quantified peak to determine if the linearity threshold is outside or within the specified tolerance Quantitation Limit Set a specified quantitation threshold tolerance value to compare with the quantitation threshold for the concentration of the quantified peak and determine if the quantitation threshold is outside or within the specified tolerance Carry over Limit Thermo Scientific Set a spe
128. ox This box displays the selected internal standard component to be used for calibration of the ISTD target compound 100 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Display Options Dialog Box in Quan Browser In the Quan Browser application use the pages of the Display Options dialog box to select Style Color Labels Axis and Normalization settings The parameters on these pages depend on whether the view in the active cell is a chromatogram or a spectrum For information about the parameters on these pages refer to the Xcalibur Qualitative Analysis User Guide or the Xcalibur Help Note The parameters on the Style Color Labels Axis and Normalization pages of the Dialog Options dialog box depend on whether the view in the active cell is a chromatogram or a spectrum The same pages for the chromatogram and spectrum views are available from the real time display view and the Qual Browser Quan Browser and Processing Setup windows To open the Display Options dialog box in the Quan Browser application 1 Open a sequence and select a file to view 2 In the chromatogram view right click and select Display Options 3 In the spectrum view right click and select Display Options Masses Dialog Box Use this dialog box to specify tolerance and precision settings for the mass data displayed in the chromatogram spectrum map and ion map plots Specify the d
129. ox to indicate that the amount was calculated by extrapolation 106 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Info Page Peak Information Dialog Box Use the Info page of the Peak Information dialog box to review the following peak properties for the current component Table 26 Info page parameters Peak Information dialog box Parameter Description Left View the left extreme of the integration baseline for the current component in minutes read only Apex View the apex point in minutes of the integration baseline for the current component read only Right View the right extreme of the integration baseline for the current component in minutes read only Height View the height of the current component peak apex in units of counts read only Area View the area of the current component peak in units of count seconds read only Baseline View the baseline height directly below the apex of the current component peak in units of counts read only Base Peak View the mass to charge ratio of the ion with the largest response in the current component peak read only Signal To Noise View the measured signal to noise ratio at the apex of the current component peak Expected RT View the expected retention time of the current component read only ISTD Response View the integrated area in units of count minutes or the height of the apex in un
130. page 40 for more information If you have identified problems with noise in the peak unresolved peaks or peak tailing change parameters on the Integration page See Setting Genesis Integration Values on page 41 for more information If baseline noise is interfering with peak identification or integration modify the settings on the Advanced page Use advanced options only if the standard options do not provide sufficiently selective detection criteria See Setting Genesis Advanced Parameter Values on page 43 for more information To view or change information on the Flags page see Setting Flags Values on page 45 for more information To save your settings as a new processing method choose File gt Export Method Quantitative Analysis User Guide 37 4 Working with Peaks Setting User Integration Settings Setting Identification Values The application uses the Identification page parameters for the following Generating a chromatogram from raw data Identifying the component within the chromatogram To set identification parameters From the User Identification Settings dialog box click the Identification tab The User Identification Settings Dialog Box opens with the Identification page displayed see Figure 17 Figure 17 Identification page User Identification Settings dialog box User Identification Settings Identification Detection Genesis Integration Genesis Advanced Flags
131. pened but before displaying any data the View Sample Types dialog box opens to prompt you to display only Standards and QC samples or All samples A Dont ask again check box is provided so that you do not have to see the dialog box again after making your initial choice To reset the display of this dialog box and all other message type dialog boxes choose Options gt Enable Warnings Table 44 View Sample Types dialog box parameters Parameter Description Viewing Options Show Standards and QCs Display only Standards and QCs in the Quan Browser grid view The data system does not display blanks and Unknowns Select one of these tabs Standards or QCs Show All Sample Types Display Standards QCs Blanks and Unknowns in the Quan Browser grid view You will be able to select from the following tabs All Standards QCs Blanks or Unknowns Don t Ask Again Decide whether you want to see the current message box or dialog box in the future For example if you always select the Show All Sample Types option and never select the Show Standards or QCs option you might want to turn off the View Sample Types dialog box To turn on the display of all message boxes and dialog boxes choose Options gt Enable Warnings 140 Quantitative Analysis User Guide Thermo Scientific ee Index A advanced integration parameters 43 120 Avalon Integration page 132 blue baselines 46 bracket types 12 Brackets Grou
132. ple are as follows e Low if the Difference is lt 0 e High if the Difference is gt 0 e Fail if the Difference is gt the user specified percentage test value To set up a stability check for a sequence run In the Sequence Setup view create a sequence that contains QC check standards at specified intervals In the Processing Setup window click the Levels tab Set up the QC table for each component as follows a Select the component in the Component list b For each QC level do the following i In the QC Level column type a name to identify the QC level ii In the Amounts column specify the amount of the target compound in the QC check standards iii In the Test column enter an acceptable difference as a percentage between the expected amount and the calculated measured amount of the target compound In the Sequence Setup window run the sequence and process or batch reprocess the data files with the Processing Method The Xcalibur data system estimates the amount of the target compound in the QC standard from a least squares fit calibration curve It then compares the measured amount of the target compound in the QC check standard to the amount specified in the QC table Quantitative Analysis User Guide 27 3 Working with the Quan Browser Results Grid Making Changes in the Results Grid Making Changes in the Results Grid In the Results grid you can change the selections in the Samp
133. plication gets peak detection calibration and quantitation information from the result file Within Quan Browser you can apply unique peak detection parameters to the chromatogram from the User Identification Settings Dialog Box This box duplicates the parameters available on the Identification and Detection pages in the Quan view of Processing Setup so you can adjust and test the effect of different values For more information about the parameters and pages for this dialog box see User Identification Settings Dialog Box on page 120 To test and change the identification and detection criteria for a component peak Review the displayed data for the selected component to determine if the results are consistent with your expectations e Are there peaks that were not found e Are neighboring peaks resolved e Are tailing peaks detected properly To modify identification and detection criteria right click the Chromatogram view and choose User Peak Detection Settings from the shortcut menu The User Identification Settings Dialog Box opens with the Identification page displayed see Figure 17 on page 38 To change the chromatogram information or adjust the retention time window change the settings on the Identification page See Setting Identification Values on page 38 for more information To change the detection method change the settings on the Detection page See Setting Detection Values on
134. ply Help The dialog box lists all the replicates used in the current bracket or group and their exclusion status Levels are listed under the following headings Level Expected Diff Exclude Shows the name for the level Displays the expected amount for the level Shows the percentage difference between measured and expected amounts Denotes excluded levels by the word Yes 2 To exclude a level click the Exclude column adjacent to the level to be excluded The application does the following e Recalculates the calibration curve without any samples using the level e Updates the corresponding Peak Status and Exclude fields in the Results grid to show that the samples are excluded e Redraws excluded data points as unfilled squares Thermo Scientific Quantitative Analysis User Guide 57 5 Working with Calibration Settings Changing the Isotope Percentage Value 3 To restore an excluded level click the Exclude column adjacent to the level to be restored on the word Yes The application does the following e Incorporates all samples using the level into the calibration and recalculates the curve e Updates corresponding Peak Status and Exclude fields in the Results grid to show that the points are now included e Redraws the included data point as a filled square 4 To save the new settings and close the dialog box click OK Changing the Isotope Percentage Value Use the Isotope page see Figure 26 t
135. ps In Use box 18 C Cal Exclusion List dialog box 57 calibration modifying parameters 51 replicates 11 calibration curve restoring a point 61 using external standard figure 7 calibration file setting 18 calibration settings Curve page 55 Levels page 58 Calibration Settings dialog box 55 93 Chromatogram view about 19 reviewing 33 working in 33 chromatogram integrating peaks manually 46 Columns command 31 component list setting 17 continuing calibration method 12 Detection page User Identification Settings dialog box 40 detection limit 5 Thermo Scientific E Exclusion List command 57 External calibration file 12 external standards considering variables for 6 7 F File menu for Quan Browser 74 file types supported 15 49 G Genesis Advanced page 43 137 Genesis Integration page 130 GoTo menu Quan Browser 77 H Help menu Quan Browser 77 ICIS Advanced page 138 ICIS Integration page 131 Identification page User Identification Settings dialog box 38 Include command 61 Include Sample Reports check box 66 Include Summary Reports check box 66 integration algorithms Avalon 132 Genesis 130 137 ICIS 131 138 integration parameters advanced 65 integration manual 46 internal standards ISTDs choosing 9 considering variables for 8 definition 7 using for quantitation 8 Quantitative Analysis User Guide 141 Index L L Levels page Calibration Settings dialog box 58 limit of
136. r Guide 3 Run the sequence to acquire the raw data files Run either one sample or a series of samples from the current sequence For more information about running samples refer to the Xcalibur Data Acquisition and Processing User Guide 4 Create a processing method Create processing methods in the Processing Setup view The data system uses a processing method to identify detect and integrate components in a chromatogram generate calibration curves quantify unknowns and produce reports The application contains several built in report templates Report templates have an xrt file extension For more information about creating a processing method refer to the Xcalibur Data Acquisition and Processing User Guide 5 Create a processing sequence by adding the processing method to the original acquisition sequence A processing sequence contains a processing method consists of a list of sample data files and includes information on sample type and calibration or QC level 2 Quantitative Analysis User Guide Thermo Scientific Thermo Scientific 1 Overview of Quantitative Analysis Acquiring and Quantitatively Processing Data 6 Process a representative raw file or the entire sequence with the processing method by using the Batch Reprocess feature in the sequence Setup view Processing a raw file produces a result file Result files have an rst file extension For instructions on batch processing a sequence refer to the
137. r Guide n A Quan Browser Reference The Quan Browser Window The Quan Browser Window The Quan Browser window consists of a title bar toolbar menu bar and status bar The workspace area contains the Results grid Components list Chromatogram view and Companion view The Chromatogram view is always displayed in the lower left portion of the window You can display the Spectrum Plot view or the Calibration Curve view in the Companion view For more information about the elements of the Quan Browser window see these topics Quan Browser Title Bar on page 73 Quan Browser Menu Bar on page 73 Quan Browser Toolbar on page 78 Quan Browser Results Grid on page 81 Quan Browser Component List on page 84 Quan Browser Chromatogram Plot View on page 84 Quan Browser Spectrum Plot View on page 86 Quan Browser Calibration Curve View on page 87 Table 1 provides brief descriptions of the workspace areas in the Quan Browser window Table 1 Quan Browser workspace Sheet 1 of 2 Area Results grid Use View the sample rows that are retrieved from the sequence or Result file You can right click this view to display a shortcut menu Use this menu to review and edit the Results grid Component list View a list of all of the components contained in the current bracket This list is in the order of user data entry in the processing method Found Not Found symbols are placed to the
138. ration Settings dialog box to review the current component type settings that you specified during method development Table 21 Levels page parameters Calibration Settings dialog box Parameter Description Cal Level View the calibration levels for the selected component Amount View the amount of target compound used for each calibration level QC Level View QC quality control level names quality control level amounts and test values Amount Test Table QC samples containing known amounts of a component can be utilized to help ensure the settings accuracy of an analysis The data system measures the quantity of the QC component in the same manner as unknown components and compares the measured quantity with a user defined expected quantity and a user defined percent test QC Level View the quality control levels for the selected component The application can accommodate up to 15 QC levels Amount View the quantity used for each QC level as defined by the user Test View a value for the acceptable difference as a percent between the known amount and the calculated measured amount of each QC level Units View the units set on the Calibration page of the Quan view in Processing Setup The application also uses the units in reports and in Quan Browser 98 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Type Page Calibration Settings Dialog Box T
139. re changed the data system recalculates all quantitation parameters including peak areas and the calibration curve Thermo Scientific Quantitative Analysis User Guide 129 A Quan Browser Reference Quan Browser Dialog Boxes Integration Page User Identification Settings Dialog Box Use the Integration page to change the current peak integration criteria for the selected component You can then test the results of the new criteria by clicking Apply or OK You can apply the new criteria to all files in the Result list by clicking Apply To All These settings are used by the Detection algorithm Depending on the peak detection algorithm that you are using one of three Integration pages is available For information about the parameters on these pages see these topics e Genesis Integration Page Parameters e ICIS Integration Page Parameters e Avalon Integration Page Parameters Genesis Integration Page Parameters The following table lists the Genesis Integration page parameters Table 38 Genesis Integration page parameters User Identification Settings dialog box Sheet 1 of 2 Parameter Description Smoothing Points Determine the degree of data smoothing to be performed on the active component peak prior to peak detection and integration The valid range is any odd value between 1 no smoothing through 15 maximum smoothing To smooth your component peak data prior to integration type a value in the Smoothing Points box
140. re of their quantity 1 Y Specify a weight value of 1 Y for all calibration data points during the least squares regression calculation of the calibration curve The data system assigns a weight value of the inverse of their response or response ratio 1 Y 2 Specify a weighting of 1 Y 2 for all calibration data points during the least squares regression calculation of the calibration curve The data system assigns a weight value of the inverse of the square of their response or response ratio 1 s 2 Specify a weighting of 1 s 2 for all calibration data points during the least squares regression calculation of the calibration curve Calibrants at a given level are weighted by the inverse of the standard deviation of their responses or response ratios For this weighting factor to be used there must be two or more replicates at each level If only one calibrant is available for any level 1 s 2 weighting cannot be used Units View the units set on the Calibration page of the Quan view of Processing Setup The units are also used in reports and in Quan Browser 94 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Flags Page Calibration Settings Dialog Box The Flags page of the Calibration Settings dialog box displays the current calibration and quantitation flags in use for the selected compound The Xcalibur data system uses these values in determining if t
141. rence Quan Browser Dialog Boxes Table 32 Reports dialog box parameters Sheet 4 of 4 Parameter Save As Description View or change the selected file export option for the sample report These are the valid export file types e None print only no exported file e Text ASCII text file txt Doc Microsoft Word file doc e HTML HTML file html PDF Adobe Acrobat file pdf e RTF rich text file rtf e XLS Microsoft Excel file xls The data system saves the exported file with the sample file name and the appropriate extension in the Data folder where result files are stored Report Template Name View the name of the summary report template to be used in processing the summary report The summary report is printed using a previously created template Double click the grid cell to open a browse dialog box so that you can select a template file Select the cell and press the F2 key to edit the box entry For additional information on how to create a report template refer to the XReport User Guide Other Controls Include Sample Report Select whether or not to print sample reports when you process the current data This option controls the printing of all of the sample reports defined in the Sample Reports box Include Summary Report Select whether or not to print summary reports when you process the current data This option controls the printing of all of the summary reports define
142. response the data system integrates each qualifier ion peak and ratios it with the quantitation peak area The application then compares this ratio with your specified target ratio If the calculated ratio is outside of the target ratio by more than your specified tolerance Window the quantitation peak is rejected e Ifyou are using Height response the data system ratios the qualifier ion peak height with that of quantitation peak The application then compares this ratio with your specified target ratio If the calculated ratio is outside of the target ratio by more than your specified tolerance Window the quantitation peak is rejected m z This column in the Qualifier Ion table displays the mass charge m z value for a qualifier ion The target ratio tolerance for this m z value is displayed in the adjacent Window table box Use other rows of the table to enter data for five qualifier ions The data system uses this data to confirm the identity of a quantitation peak by comparing the relative responses of qualifier ion and quantitation peaks with predetermined values Target Ratio This column in the Qualifier Ion table displays the Target Ratio value for a qualifier ion The m z value and target ratio tolerance for the qualifier ion are given in the adjacent m z and Window table boxes Use other rows of the table to enter data for five qualifier ions The data system uses this data to confirm the identity of a quantitatio
143. ria do not provide the desired results You can then test the results of the new criteria by clicking Apply or OK You can apply the new criteria to all files in the Result list by clicking Apply To All Advanced parameters used for the detection and integration of peaks are less often used but can provide adequate peak detection with the default parameters You can set advanced parameters for the Genesis and ICIS algorithms For more information see these topics e Genesis Advanced Page Parameters e ICIS Advanced Page Parameters 136 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Genesis Advanced Page Parameters Table 41 Genesis Advanced page parameters User Identification Settings dialog box Parameter Report Noise As Description RMS Select this option to calculate noise as RMS Peak To Peak Select this option to calculate noise as peak to peak Manual Noise Region Specify the region of the chromatogram that the Xcalibur data system uses to determine noise You can click J and drag the cursor horizontally across the region of the chromatogram that you want to select as the noise region or type the retention time RT in the RT Range box The data system marks the region with a red baseline RT Range Specify the retention time RT range The RT range should be within the chromatogram range You can click and drag the cursor horizontally acro
144. rmation drugx User Peak Detection Settings Apply unique peak detection parameters to the chromatogram The tabbed pages on this dialog box differ for GC and LC processing methods These differences are described in the appropriate sections The type of processing used to create the result file comes from the processing method also stored within the result file and are not available for changes within Quan Browser Display Options Place labels such as retention time scan number base peak signal to noise flags area and height on your chromatograms This menu command is not active if no chromatogram data is displayed in the Chromatogram Plot view 84 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference The Quan Browser Window Table 11 Chromatogram Plot view shortcut commands Sheet 2 of 2 Command Description Manually Add Peak Manually place a baseline on the Chromatogram Plot and set the current integration method for the current component to Manual Integration The Xcalibur data system changes the entry in the Integration Type box of the Results grid view If no peak has been detected for the currently selected compound there is no integration baseline on the chromatogram plot for you to manually adjust Set Peak To Not Found Tag the current peak as Not Found do another peak search possibly with new integration Status parameters or do a manual integration to restore the peak
145. rocessing method associated with the currently loaded sequence To select a component of interest select the component name from the list All Quan Browser views are automatically updated to reflect the settings and data for the selected component The component list provides the only method of selecting a component of interest Figure 31 Component list for the drugx example set D4 Quan Browser Chromatogram Plot View Table 11 Chromatogram Plot view shortcut commands Sheet 1 of 2 Command Method Settings Description Reset the integration method to those specified in the original method stored within the result file User Settings Manual Integration Apply a set of user defined peak detection parameters to the integration of this peak See Setting User Integration Settings on page 37 for more information If active this command indicates that the integration baseline has been adjusted by dragging the control boxes on the plot This command is inactive until you adjust the baseline by using the graphical dragging operation at least once Show Peak Info View peak information in a read only format The information displayed reflects the settings for the currently displayed chromatographic peak Select other compounds or other result files to get detailed information on the found peak To close the dialog box click Close The compound identification name is displayed in the title bar For example Peak Info
146. roup of samples These are the sample files that you select from for processing and for printing a report Sample Type View the sample type of the raw file displayed to the left on the same row Selected Samples Raw File View or change the raw files that have been selected from the Sample Choices box These are the sample files to be processed so that you can print a report Sample Type View the sample type of the raw file displayed to the left on the same row Buttons Add Add files selected in the Sample Choices box to the Selected Samples box Remove Return files selected in the Selected Choices box back to the Sample Choices box Add All Add all of the files in the Sample Choices box to the Selected Samples box Remove All Return all files in the Selected Choices box back to the Sample Choices box Thermo Scientific Quantitative Analysis User Guide 119 A Quan Browser Reference Quan Browser Dialog Boxes User Identification Settings Dialog Box Use the User Identification Settings dialog box to select and test mass scan filter relative peak height threshold peak identification and peak integration settings If these standard options do not provide the desired results Quan Browser also provides advanced options using the Advanced page The pages that are displayed depend on whether you are currently using the Genesis the ICIS or the Avalon peak detection algorithm For information about the identification detec
147. rtcut menu Figure 8 Chromatogram view and shortcut menu steroids02 Method Setting 212811336 11 20 31 AI Method Settings RT 0 18 1 18 SM 7G User Setti 400 RT 0 68 NL 5 01E5 miz Manual Integration 266 6 267 6 80 308 6 309 6 F c Full Show Peak Info 8 ms2 363 20 cid By 150 00 375 00 MS 5 L 60 aap ar User Peak Detection Settings a lt y 3 j Display Options as e Manually Add Peak Set Peak to Not Found Status 0 52 0 95 0 Update Expected Retention Time gt 0 2 0 4 0 6 0 8 1 0 Time min Reset Scaling Thermo Scientific Quantitative Analysis User Guide 19 2 Quan Browser Overview Quan Browser Window Overview When a filter is stored within the embedded processing method for the current compound the application applies it to the chromatogram Adjust the chromatogram plot using the Zoom menu commands or buttons on the toolbar The type of integration used appears in the Results grid but can be overridden The three types are Method Settings User Integration and Manual Integration Change the Integration method by using the User Identification Settings dialog box or by manually integrating a peak For more information see Setting User Integration Settings on page 37 and Integrating Chromatogram Peaks Manually on page 46 The Companion view is located to the right of the Chromatogram Plot view its companion in the lower right corner of the Quan Browser window In the c
148. s View the name of the report template to be used in processing the data and generating the reports indicated by Yes in each row The sample report is printed using a previously created template Double click the grid cell to open a browse dialog box so that you can select a template file Select the cell and press the lt F2 gt key to edit the box entry For additional information on how to create a report template refer to the XReport User Guide Summary Reports Select summary reports from displayed boxes The report to be printed is defined in the Report Template Name box You can enable the processing of a summary report by entering Yes in the Enabled box or turn off the processing by clearing the box Enabled Thermo Scientific Specify whether or not the summary reports are processed using the templates that appear in the Report Template Name boxes For example if the Enable box in the row displays Yes then the data system prints the summary report defined by the report template in the row If the Enabled box in the row is clear then the application does not print this summary report When you select an Enabled box a check box control appears If you select this check box and you click another cell the application displays the word Yes to indicate that the report is enabled If you do not select the check box the cell remains blank when you click another cell Quantitative Analysis User Guide 115 A Quan Browser Refe
149. s More Flags Suitability and Spectrum Review the read only results 8 To review the other spectrum search results choose the Show Peak Info Candidate N command from the Chromatogram Plot shortcut menu The Xcalibur system opens the Peak Information dialog box with the following title Peak Information Component Spectrum Candidate This dialog box displays the following pages Info Chro and Spectrum The Chro page displays a total ion current plot of the Spectrum Candidate The view is centered around the mass of interest and has the width used by the component peak display Review the read only results Repeat this step for other candidates of interest 9 To close the Peak Information dialog box click Close To enter the spectrum search settings in the Processing Setup window 1 In the Processing Setup window open the processing method that you want to modify 2 Open the Detection page of the Quan view 3 In the Peak Detection area select the Spectrum option 4 Set up the search criteria as described in the Xcalibur Data Acquisition and Processing User Guide 50 Quantitative Analysis User Guide Thermo Scientific ee lt a Working with Calibration Settings When you first open a sequence in the Quan Browser window the Xcalibur data system performs peak detection calibration and quantitation according to the settings in the associated processing method Within the Quan Browser application you can chang
150. s User Guide 125 A Quan Browser Reference Quan Browser Dialog Boxes Table 37 Detection page parameters User Identification Settings dialog box Sheet 2 of 5 Parameter Reverse Description Set a threshold value for Reverse comparisons between the reference spectrum and candidates in the chromatogram A Reverse search ignores any peaks in the unknown that are not in the reference spectrum in the peak identification table The match is scored on a scale of 0 to 999 A perfect match results in a score of 999 As a general guide 900 or greater is an excellent match 800 to 900 a good match 700 to 800 a fair match Less than 600 is a poor match A spectrum with many peaks tends to score more highly in a Reverse match than a Forward match Match Set a threshold value for Match comparisons between the reference spectrum and candidates in the chromatogram Match is scored on a scale of 0 to 999 The Match algorithm is a complex probability factor based on the differences between the Forward factors of all the candidates If one candidate has a Forward matching factor of 900 and the next best is only 300 the probability of the component being correctly identified is high and so the Match factor is scored highly for the first candidate If the Forward factors for all the candidates are similar whether high or low the Match factor is low Highest Peak This option is available for both LC MS and GC MS data Use the highest peak i
151. s and Exclude field in the Results grid and Exclusion List to show that the point is now included e Redraws the included data point as a filled square Include or exclude samples that are shared between brackets Their status is unique to the bracket For example excluding a shared sample in bracket 1 has no effect on the inclusion status in bracket 2 Quantitative Analysis User Guide 61 ee i Viewing Results and Generating Reports Follow these procedures to review the quantitative results and generate reports Contents e Reviewing the Results of the Quantitative Analysis Generating Reports Reviewing the Results of the Quantitative Analysis To review and rework results before you print reports 1 Open the Quan Browser window see Starting Quan Browser on page 15 The Open dialog box opens so that you can select a file to review and rework You can select a sequence list file sld extension a result file rst extension or a Quan Browser file xqn extension 2 Select the file and click OK The View Sample Types dialog box opens 3 Select either the Show Standard and QC sample types or Show All sample types option Click OK The Quan Browser window opens with the Results grid view Component list view Spectrum Plot view and Calibration or Spectrum Plot view 4 In the Component list view select the target component that you want to review 5 To display calibration standards results click t
152. s software package that you can use to acquire data specifically for analytes of interest to perform confirmatory library searches and to determine the concentration of analytes in samples The Xcalibur data system interfaces with the XReport reporting package to print individual sample reports and sequence summary reports for analyses For more information on XReport see the XReport User Guide Quan Browser is a powerful and versatile utility for reviewing and reworking the following e Component peak identification and integration criteria e Standards QCs blanks and unknowns e Calibration curves for quantitation standards After making any changes save the new results with an audit trail describing the reason for the change Quan Browser incorporates a calibration curve display peak integration and results view where you can do the following e Process quantitation sequences e Interactively edit processing parameters and audit the changes e Create new files that keep track of processing results for individual raw files and include a copy of the method used to generate the results Result files changed using Quan Browser do not affect the original processing method This chapter describes some of the basic principles and terminology of quantitation and provides a brief overview of quantitation with the Xcalibur data system Contents e Acquiring and Quantitatively Processing Data e Integrating and Identifying Chromatograp
153. samples in the Results grid Tom Instrument Setup Open the Instrument Setup window Processing Setup Open the Processing Setup window Qual Browser Open the Qual Browser window Library Browser Open the Library Browser window Xcalibur Home Page Open the Home Page window if it is closed or display the Home Page window if it is already open This command closes the Instrument Setup window so that all instrument setup methods are closed when samples that use these methods are run from the Home Page window 5 Quan Browser Help 80 Quantitative Analysis User Guide View Help for Quan Browser Thermo Scientific A Quan Browser Reference The Quan Browser Window Quan Browser Results Grid The Results grid displays sequence or result sample data entries Each row defines a result file and its associated parameters Selecting any cell within the Results grid causes the additional views to refresh using data from the newly selected sequence row The Xcalibur data system validates any changes made to the sequence row and updates displays automatically Figure 30 Results grid view Sample Sample Integration File Name Type CAS Type 1 drugx_01 Standard Method Settings 2 drugx_02 Standard Method Settings 3 drugx_03 Standard Method Settings 4 dugs_04 Standard Method Settings drugx_05 Standard Method Settings 6 dugx_06 Standard Method Settings Z drugx_O7 Sta
154. se Region Rise Percentage Valley S N Peak S N Cutoff Baseline Noise Tolerance Min Number of Scans in Baseline Number of Background Scans Cancel Apply Apply Toal Help 2 To calculate noise as RMS select the RMS option 3 To have the data system calculate noise as peak to peak select the Peak to Peak option 4 To specify the region of the chromatogram that the Xcalibur data system uses to determine noise retention time type a retention time RT in the RT Range box You can also click in the toolbar and drag the cursor horizontally across the region of the chromatogram that you want to select as the noise region The data system automatically fills the RT Range box with the retention time value 5 To enter a value in a percentage that specifies how high the peak trace can rise above the baseline after passing through a minimum before or after the peak type a value from 0 1 to 500 0 in the Rise Percentage box If the trace exceeds this value the application applies valley detection peak integration criteria This test is applied to both the left and right edge of the peak This criteria is useful for integrating peaks with long tails To apply the new peak detection criteria click OK Quantitative Analysis User Guide 43 4 Working with Peaks Setting User Integration Settings 10 11 To change the valley detection signal to noise criteria that the Xcalibur data system uses for
155. selected sample reports ee RE 78 Print Enabled Summary Reports Quantitative Analysis User Guide Print all active summary reports Thermo Scientific A Quan Browser Reference The Quan Browser Window Table 8 Quan Browser toolbar buttons Sheet 2 of 3 Description Show Calibration Curve View the Calibration Curve view in the lower right corner of the Quan Browser window Show Spectrum Plot View the Spectrum Plot view in the lower right corner of the Quan Browser window aie Reports Dialog Select and enable previously created report templates and other options for sample reports and summary reports cre Show Large Toolbar View either the large or small Quan Browser toolbar The Xcalibur data system displays the large toolbar when you select the check box to the left of the command The system displays the small toolbar when you clear the check mark to the left of the Show Large Toolbar command Zoom 4 Zoom In Y To show more detail zoom in on the Y axis by a factor of two 2 from the current baseline J3 Zoom Out Y To show more data zoom out on the Y axis by a factor of two 2 Ei Auto Range View the chromatogram which is normalized from the minimum to the maximum signal This zoom feature is recommended for PDA and UV data l Normalize Normalize the intensity scale of the data display to a fixed range on the Y axis for example from 0 25 to 0 100
156. ser Results Grid Reviewing and Reworking the Results Grid c Browse to and select the file of interest and click Open When you open a bracketed sequence the Bracket in Use list is active and displays the available brackets A bracket sequence opens with bracket 1 displayed When you open an unbracketed sequence the Group in Use list is active and displays groups broken up logically For more information about brackets and groups see Bracket Group In Use List on page 91 When you open a result file the application creates a one row sequence 2 Inspect the sequence If the sequence contains more than one bracket inspect each bracket Verify that the correct samples are listed in the Results grid Ensure that each sample in the sequence is properly associated with its sample type and that the appropriate levels are associated with the standards and QC samples Check the status in the Peak Status column 3 To change the sample type of a sample in the Results grid click the Sample Type column to display the sample type list and then select the new sample type 4 To change the level for a standard or QC sample type click the Level column to display the Level list and then select a new Level from the list 5 To add or remove samples see Adding and Removing Samples on page 28 6 To hide or show columns see Hiding or Displaying Columns on page 31 7 To change the sort order of the sequence rows see Changing th
157. shortcut menu The data system removes the blue baseline to the component peak reduces the Area to 0 and changes the Peak Status to Not Found To change the status of a peak from Not Found to Found Added optional right click in the Chromatogram Plot view and choose Manually Add Peak from the shortcut menu The application adds a blue baseline to the component peak integrates the peak displays the Area in the Grid Results view and changes the Integration Type to Manual Integration Edit baseline integration criteria of a Found or Added peak optional a Click the left or right square editable handle on the blue baseline of the selected peak The data system changes the cursor to a a b Drag the handle to define a new location of the left or right peak limit Repeat this procedure for the opposite side of the peak if required The Xcalibur data system automatically recalculates the results for the component and displays them in the Result grid view If the sample was a standard the application replots the data and redraws the calibration curve in the Calibration Curve view Quantitative Analysis User Guide 47 4 Working with Peaks Using the Spectrum Plot Companion View Using the Spectrum Plot Companion View The Spectrum Plot companion view displays a spectrum from the current chromatogram in the Chromatogram view View spectra from the apex left peak edge or right peak edge using commands from the shortcut menu
158. spect to acquisition date and time It then examines the sequence and starts a new group whenever it encounters a standard The group ends at the nonstandard sample that immediately precedes the next standard found The first group always starts with the first sample even if it is not a standard The last group always ends with the last sample Further a Std Clear always starts a new group even if no intervening nonstandard sample has been found following one or more Std Updates The Xcalibur data system forms additional logical groups if different named calibration files have been specified in the Cal File entries of the sequence Each cal file entry causes a new group to be formed Because using multiple named calibration files is not typical their use is not considered any further in this document but should be deducible from the discussions on groups 12 Quantitative Analysis User Guide Thermo Scientific Thermo Scientific 2 Quan Browser Overview Understanding How Quan Browser Works As the Quan Browser application processes each group it quantifies samples against the current calibration curve The application processes each standard that it encounters by either replacing sample type set to Std Clear or adding to sample type set to Std Update the calibration replicate list generating a new calibration curve Quan Browser processing closely emulates that of batch processing either batch processing directly after acquisition or
159. ss the region of the chromatogram that you want to select as the noise region or type a value in the RT Range box The data system marks the region with a red baseline Rise Percentage View or adjust the percentage that the peak trace can rise above the baseline after passing through a minimum before or after the peak If the trace exceeds this value the data system applies valley detection peak integration criteria This test is applied to both the left and right edge of the peak This criteria is useful for integrating peaks with long tails The valid range is 0 1 to 500 0 To change the rise percentage type a value in the Rise Percentage box Click OK to apply the new peak detection criteria Valley S N Peaks S N Cutoff View or adjust the signal to noise criteria that the data system uses for valley detection The valid range is 1 0 to 100 0 To change the valley detection signal to noise criteria type a value in the Valley S N box Click OK to apply the new peak detection criteria View or adjust the signal to noise below which the data system defines the peak edge For example if the signal to noise at the apex is 500 and the Peak S N Cutoff value is 200 the application defines the right and left edges of the peak when the S N reaches a value less than 200 The valid range is 50 0 to 10000 0 Baseline Noise View or adjust a value that controls how the baseline is drawn in the noise data The higher Tolerance the b
160. subsequently as a batch process operation If the Quan Browser application cannot find or open a specified calibration file the Xcalibur data system displays this message Cal File Unavailable Using Embedded Calibration in the Calibration File edit box The Quan Browser application takes replicate data from the data stored in the result file In most cases this data is identical to the data contained within the original calibration file Once the Quan Browser application has set up the groups they are independent and are effectively treated as brackets In other words changes in one group do not affect any other group unlike in batch processing where subsequent groups might well be affected The following list illustrates the procedure for a single named calibration file Sample 1 Unknown Group 1 start Sample 2 Unknown Group 1 end Sample 3 Std Clear Group 2 start Sample 4 Unknown Group 2 end Sample 5 Std Update Group 3 start Sample 6 Std Update Sample 7 Unknown Sample 8 Unknown Sample 9 Blank Sample 10 QC Group 3 end Sample 11 Std Update Group 4 start end Sample 12 Std Clear Group 5 start Sample 13 Blank Group 5 end Quantitative Analysis User Guide 13 2 Quan Browser Overview Understanding How Quan Browser Works Open Bracket Sequence For an open bracket sequence Open bracket type the Quan Browser application creates a replicate list directly from all standard samples in the sequence without using any calibra
161. sults grid changing the sort order 32 column headings 31 63 displaying columns 31 editing a sequence 25 hiding columns 31 working in 18 S saving changes 26 scaling resetting 48 Select Report Samples dialog box 67 Select Samples button 66 Set Sorting Order command 32 Show Calibration Curve command 48 Show Peak Info command 35 Show Standards and QC commands 15 sort order 32 Spectrum at Peak Apex command 48 Spectrum at Peak Left Edge command 48 Spectrum at Peak Right Edge command 48 Spectrum Plot view 48 spreadsheets exporting the Results grid to 23 standard clear 12 update 12 system suitability results 36 Thermo Scientific T target component 5 U unbracketed sequence 12 upper quantitation limit 5 User Identification Settings dialog box Detection page 40 ICIS Advanced page 43 ICIS Integration page 41 Identification page 38 User Peak Detection Settings command 37 V variables discussion of quantitation with external standards 6 quantitation with internal standards 8 View menu Quan Browser 75 View Sample Types dialog box 15 View Spectrum Plot command 48 W Warning dialog box 15 working in the Results grid 18 X Xcal files 12 Xcalibur data system acquiring and processing data with overview 2 quantitative analysis procedure 2 Xcalibur Quan file saving 21 Z Zoom menu Quan Browser 76 Thermo Scientific Quantitative Analysis User Guide Index T 143
162. t of Quantitation Linearity Limit View or change the current value for the linearity limit The data system sets the Limit of Linearity LL Flag in the result file if the peak concentration is greater than the entered Linearity Limit Linearity Limit 10 percent is the calibration curve s X axis upper value default when displayed in the Quan Browser window The user can then go on to change this X axis range Carry Over Limit Thermo Scientific View or change the current value for the carryover limit The data system sets the Carry Over Limit COL flag in the result file if the peak concentration is greater than the entered Carry Over Limit Quantitative Analysis User Guide 95 A Quan Browser Reference Quan Browser Dialog Boxes Isotope Page Calibration Settings Dialog Box Use the Isotope page of the Calibration Settings dialog box to correct for an impurity in the internal standard compound that elutes at the same time as the target compound or to correct for an impurity in the target compound that elutes at the same time as the internal standard Table 20 Isotope page parameters Calibration Settings dialog box Sheet 1 of 2 Parameter Description Contribution of ISTD To Target View the ratio C d SE ISTD impurity ISTD pure Where ISTD impurity is an impurity compound in the internal standard reagent that elutes at the same time as the target compound ISTD pure is the pure interna
163. t value type a new width in the Expected Width box 130 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Table 38 Genesis Integration page parameters User Identification Settings dialog box Sheet 2 of 2 Parameter Description Constrain Peak Width Limit the peak width of a component during peak integration of a chromatogram You can then set values that control when peak integration is turned on and off by specifying a peak height threshold and a tailing factor To constrain a peak width select the Constrain Peak Width check box The Peak Height box and the Tailing Factor box are activated Peak Ht View or adjust the percent of the total peak height 100 that a signal needs to be above the baseline before integration is turned on or off This box is active only when the Constrain Peak Width check box is selected The valid range is 0 0 to 100 0 To enter this height type the appropriate value in the Peak Ht box Tailing Factor View or adjust a factor that controls how the Xcalibur data system integrates the tail of a peak This tailing factor is the maximum ratio of the trailing edge to the leading side of a constrained peak This box is active only when the Constrain Peak Width box is selected The valid range is 0 5 through 9 0 ICIS Integration Page Parameters The following table lists the ICIS Integration page parameters Table 39 ICIS Integration page paramet
164. template that appears in the Report Template Name box of the same row To print an Other sample report the data system must display Yes in the Enabled box in the same row For example if the Other box displays Yes and the Enable box in the same row displays Yes then the application prints the Other sample report However if the Enabled box in the row is clear then the application does not print the Other sample report When you select an Other box a check box control appears If you select this check box and you click another cell the application displays the word Yes to indicate that the report is enabled for Other samples If you do not select the check box the cell remains blank when you click another cell 114 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Table 32 Reports dialog box parameters Sheet 3 of 4 Parameter Save As Description View the selected file export option for the sample report These are the valid export file types e None print only no exported file e Text ASCII text file txt Doc Microsoft Word file doc e HTML HTML file html PDF Adobe Acrobat file pdf e RTF rich text file rtf e XLS Microsoft Excel file xls The data system saves the exported file with the sample file name and the appropriate extension in the Data folder where result files are stored Report Template Name Summary Report
165. tention time reference component and whether it was used correctly by the processing method If there was a retention time reference then the check box indicates whether or not the retention time reference peak was found The data system indicates it found the peak by selecting the check box The application indicates it looked for the peak but did not find it by leaving the box blank If there is no retention time reference then the check box is selected because there is no correction to be made Response OK View whether or not a response factor was calculated The data system selects the check box to indicate that it found the peak and the peak s internal standard and correctly calculated the response ratio Response Low View whether the calculated amount for the peak was less than the lowest specified standard amount of the component in the calibration curve In this case the calculated amount has been determined by extrapolation from the lowest level The data system selects the check box to indicate that the amount was calculated by extrapolation If you force or include the origin the application defines the lowest level to be 0 0 Response High View whether or not the calculated amount for the peak was greater than the highest specified standard amount of the component in the calibration curve In this case the calculated amount has been determined by extrapolation from the highest level The data system selects the check b
166. the Std box displays Yes and the Enable box in the same row displays Yes then the data system prints the QCs sample report However if the Enabled box in the row is clear then the application does not print the QCs sample report When you select a QCs box a check box control appears If you select this check box and you click another cell the data system displays the word Yes to indicate that the report is enabled for QC samples If you do not select the check box the cell remains blank when you click another cell Unks View whether or not the Unknowns Unks sample reports marked with Yes are processed using the report template that appears in the Report Template Name box of the same row To print an Unknowns sample report the data system must display Yes in the Enabled box in the same row For example if the Unks box displays Yes and the Enable box in the same row displays Yes then the application prints the Unks sample report However if the Enabled box in the row is clear then the application does not print the Unks sample report When you select an Unks box a check box control appears If you select this check box and you click another cell the data system displays the word Yes to indicate that the report is enabled for Unk samples If you do not select the check box the cell remains blank when you click another cell Other View whether or not the Other sample reports marked with Yes are processed using the report
167. the current browser session Save All Export Method Update all result files with the current information Each result file comes from a results row in the Results grid Since each row can use method user or manual integration and the result file can contain only one method the currently selected method for that row is used when creating the result file The embedded processing method is flagged as modified This means that each result file can potentially contain different embedded processing methods If read back into Quan Browser the first sample s embedded processing method is used for the method settings of the entire bracket When read in each sample that has a modified processing method is set in the User integration mode and it is up to the operator to reintegrate and quantitate using a common method if desired Export the processing method of the currently selected row If no processing method is available the data system uses the original processing method Export Data To Excel Short Report Create a short Microsoft Excel report The short report displays the data contained in the current bracket and contains the same information that is available in each Results grid row Long Report Create a long Microsoft Excel report The data system reports on the data contained in the current bracket and contains the same information that is available in each Results grid row as well as other information that is not
168. the current qualifier ion did not pass the Ion Coelution test Absolute Window View the calculated Absolute Window Percentage that Xcalibur calculated during the Ion Ratio test read only The application does not display this box if the current qualifier ion did not pass the Ion Coelution test No Peak Page Peak Information Dialog Box If the Xcalibur data system does not find a peak for the component only the No Peak page is displayed with the following message No Peak Found Cannot show Peak Info Spectrum Page Peak Information Dialog Box This read only page displays the spectrum of the current peak at the apex retention time You cannot make adjustments to this display 110 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Suitability Page Peak Information Dialog Box The Suitability page of the of the Peak Information dialog box displays the results of specific tests that might have been performed as specified by the processing method on the component peak to determine its suitability to be considered a valid peak Each test is listed with the result of that test displayed to the right There are three possible responses for each test Passed Failed and Not Tested Table 30 Suitability page parameters Peak Information dialog box Parameter Suitability Flags Description Symmetrical View the results of the Symmetrical test as Passed
169. the signal reaches the baseline independent of the value set for the expected peak width To specify a limit for the peak width of a component during peak integration of a chromatogram select the Constrain Peak Width check box You can then set values that control when peak integration is turned on and off by specifying a peak height threshold and a tailing factor To adjust the percent of the total peak height 100 that a signal must be above the baseline before integration is turned on or off type a value in the Peak Ht box To change how the data system integrates the tail of a peak type a value between 0 5 and 9 0 in the Tailing Factor box This tailing factor is the maximum ratio of the trailing edge to the leading side of a constrained peak To save your changes click OK 42 Quantitative Analysis User Guide Thermo Scientific 4 Working with Peaks Setting User Integration Settings Setting Genesis Advanced Parameter Values Thermo Scientific The Xcalibur data system applies the Genesis Advanced page parameters see Figure 20 during Genesis peak detection and integration To set Genesis advanced parameters 1 From the User Identification Settings dialog box click the Genesis Advanced tab Figure 20 Genesis Advanced page User Identification Settings dialog box User Identification Settings Identification Detection Genesis Integration Genesis Advanced Flags Report Noise As C RMS Manual Noi
170. the units used for the internal standard amount or concentration for example ng or Thermo Scientific pg mL This box becomes active when you select the ISTD component type Use the Type page for a Target component to change the Internal Standard ISTD that you specified on the Calibration page in the Quan view of the Processing Setup window The data system selects the Target Compound option and the ISTD option which are read only Quantitative Analysis User Guide 99 A Quan Browser Reference Quan Browser Dialog Boxes Table 23 Target component parameters Parameter Description Component Type The data system recognizes two component types for component calibration purposes Target Compound components and Internal Standards ISTD components Components of interest are initially defined selected and characterized using the Calibration page in the Quan view of the Processing Setup window These parameters can then be revised using the Type page of the Calibration Settings dialog box available in the Quan Browser window Target Compound Specify that the selected component is a target compound This button is only active if you have defined at least one component as an internal standard and selected another component as Component Type Target Compound ISTD Specify that the selected component is an internal standard The ISTD option is unavailable if you have selected the External Standard option in the Calibration Options dialog b
171. ting Component Types in the Processing Setup Window 54 In the Quan Browser window you cannot change the component type or the internal standard component associated with a target compound To change these calibration settings you must modify the processing method in the Quan view of the Processing Setup window and then batch reprocess the sample sequence in the Sequence Setup view R kod I To define the type for a component From the Quan view of the Processing Setup window click the Calibration tab The Calibration page opens To select a component click a component in the Component list located at the far right of the Processing Setup window To define the type of the selected component select the Target Compound option or the ISTD option When creating an internal standard method you must define at least one component to be an ISTD before you can define any other components as target compounds Figure 23 Calibration page Identification Detection Calibration Levels System Suitability Component type Target compounds Cc Target compound ISTD methyltestosterone X Isotope C ISTD Weighting Calibration curve Linear Equal Cin Units Ng C 172 Origin roo CaN C Ignore Response CAN Force Area 1 82 Include Height Lx Save As Default Flaas Help e Ifyou select the Target Compound option the Target Compounds area becomes active Go to step 4 e Ifyou select the
172. tion data embedded in result files When you open a single result file in the Quan Browser application the application treats it as a sequence with only one entry and lists the sample type as Unknown To show the calibration curve used to quantitate the sample the application creates the replicate list from the embedded information Nonoverlapping Bracket Sequence For a non overlapping bracket sequence Non Overlapping bracket type the Quan Browser application creates a separate replicate list for each bracket The application creates each replicate list directly from all standard samples in the bracket without using any calibration data embedded in result files Overlapping Bracket Sequence For an overlapping bracket sequence Overlapping bracket type the Quan Browser application creates a separate replicate list for each bracket The application creates each replicate list directly from all standard samples in the bracket without using any calibration data embedded in result files Exceptions occur for shared standard samples between brackets When a standard that is shared undergoes a change that change is reflected in all brackets that contain that standard When a shared standard sample is deleted the application deletes the standard sample in all brackets that contain that standard sample and adjusts the replicate lists for all brackets When you add a sample as a standard to any bracket the application adds it to the replicate
173. tion integration advanced and flag parameters see these topics e Identification Page User Identification Settings Dialog Box on page 121 e Detection Page User Identification Settings on page 125 e Integration Page User Identification Settings Dialog Box on page 130 Genesis Integration Page Parameters on page 130 ICIS Integration Page Parameters on page 131 Avalon Integration Page Parameters on page 132 e Advanced Page User Identification Settings Dialog Box on page 136 Genesis Advanced Page Parameters on page 137 ICIS Advanced Page Parameters on page 138 e Flags Page User Identification Settings Dialog Box on page 139 120 Quantitative Analysis User Guide Thermo Scientific A Quan Browser Reference Quan Browser Dialog Boxes Identification Page User Identification Settings Dialog Box Use the Identification page to change the current component name mass range scan filter and retention time for the selected component You can then test the results of the new settings by clicking Apply or OK You can apply the new settings to all files in the Result grid view by clicking Apply To All The Identification page helps to narrow the search parameters and to set filters so that the peak detection algorithms have an easier time of locating the peaks The compound of interest is displayed in the Name box This is a read only field To select a different
174. to the target compound through the extraction cleanup and analytical processes You can also add the ISTD component as the last step of sample preparation prior to the sample s use to compensate for fluctuations in the reproducibility of the sample injection For the internal standard calibration method the Xcalibur data system constructs a calibration curve from a set of standard solutions that contain a range of concentrations for the target compounds and a fixed concentration for each internal standard compound For each target compound the data system plots the detector response ratios for the target compound and its associated internal standard compound as a function of the corresponding target compound concentration The data system then determines the concentration of the target compounds in the unknown samples by using the calibration curve Figure 4 shows the interpolation of the target compound amount in an unknown from calibration curve constructed using the internal standard method Figure 4 Calibration curve generated by using the internal standard method 14 Response ratio for unknown sample 1 25 ra p d 5 Sy Ane a ENE Er l Se I aS 0 8 4 zg l ae ae 2 i Amount in D unknown cc 0 2 4 l va 0 0 r r i r 0 0 0 5 1 0 1 5 2 0 2 5 Amount of target compound Thermo Scientific Thermo Scientific 1 Overview of Quantitative Analysis Integrating and Identifying Chromatographic Peaks The cali
175. to this group until the next standard after the first nonstandard is found There are two deviations from this rule The first exception is that the first group does not have to start with a standard The first sample by definition begins the first group The second exception is that any Std Clear begins another group even if it immediately follows a Std Update Quantitative Analysis User Guide 91 A Quan Browser Reference Quan Browser Dialog Boxes Cal Exclusion List Dialog Box The Cal Exclusion List dialog box displays a list of all the replicates used in creating the current calibration curve In some cases there might appear be more entries in this list than are displayed in the calibration curve Typically this is because some points lie on top of each other and cannot be seen on the plot of the calibration points If this is the case it is impossible to change the exclusion status of all the overlapping points by right clicking the graphed point The Xcalibur data system sees the first point in its list that meets the position requirement and changes its status The application never sees the other data points All overlapping points can be included or excluded in the Results grid if they are listed there However when using external calibration files these data points might not have corresponding result rows in Quan Browser These replicates data points are accessible using the Cal Exclusion List dialog box The title bar of the Cal
176. tors However for some types of analyses this method cannot achieve the highest level of precision and accuracy Depending on the instrumentation variations in analyte and solution stability injection reproducibility and matrix interference can lead to lower precision levels in the external standard method than in the internal standard method 6 Quantitative Analysis User Guide Thermo Scientific 1 Overview of Quantitative Analysis Integrating and Identifying Chromatographic Peaks Figure 3 Calibration curve generated by using an external standard Response for unknown sample 500000 g D gt Qa 400000 O E S es pee pas rang ras Pe Pens Sey E E E Se E A EEEE EE E Se ey S I i 300000 l Ss l 200000 4 Amount in P j unknown sample cc l 100000 l l I 0 T T T T o 20 40 60 80 1 Amount of Target Compound In general the external standard calibration is an effective quantitation technique however if one or more of the following problems exist consider using the internal standard calibration technique instead Lack of injection reproducibility e Changes in analyte solution volume e Matrix and coeluter interference both suppression and enhancement e System instability e Variations in the source conditions Using Internal Standards Thermo Scientific An internal standard ISTD is a component that is added to a sample to act as a response reference for one or more
177. tup Open the Processing Setup window m Qual Browser Open the Qual Browser window chal 7 Library Browser Open the Library Browser window ic Xcalibur Home Page Open the Home Page window Help Menu Table 7 Help menu commands Command Quan Browser Help Description Open Xcalibur Help and view Help for the Quan Browser window Xcalibur Help Open Xcalibur Help Glossary Open the glossary How To Use Help Open Help that describes how to use the Help viewer About Quan Browser Thermo Scientific Open the About Quan Browser dialog box This dialog box displays the installed version number of the Quan Browser program and the Thermo Fisher Scientific copyright notice Quantitative Analysis User Guide 77 A Quan Browser Reference The Quan Browser Window Quan Browser Toolbar When you install the Xcalibur data system the Quan Browser toolbar displays these buttons Alma t o sfF Klwlole MIE 2 To add or remove buttons use the Customize Toolbar dialog box For information about the Customize Toolbar dialog box refer to the Xcalibur Getting Started Guide or the data system Help Table 8 describes the buttons by category that can appear on the Quan Browser toolbar Table 8 Quan Browser toolbar buttons Sheet 1 of 3 Button File Description Open Open previously saved data files The Xcalibur data system supports these file types sequence sld result rst and Quan Browser xqn
178. use the information that is stored in the raw files for these parameters keep the Use Sample Name and Comment from Selected RawFile s check box selected When you add the sample or samples to the Results grid the data system populates the Sample Name and Comments fields for the added samples with the information embedded in the raw files or e To change the Sample Names and Comments settings for the sample or samples that you are adding clear the Use Sample Name and Comment from Selected RawFile s check box Then type the appropriate text in the Sample Name and Comments boxes When you add the sample or samples to the Results grid the data system populates the Sample Name and Comments fields with your text entries When you add multiple samples at a time the data system uses the same Sample Name and Comment for all of the added samples 6 For the remaining parameters in the Add Samples dialog box do the following e Inthe Sample Type list select the appropriate sample type Note If the processing method associated with the selected sample in the Results grid does not specify any QC levels you cannot select the QC sample type e In the Levels list select the appropriate level for standard or QC sample types e In the Sample ID box type an appropriate sample ID e Inthe ISTD Corr Amt box type a value from 0 to 10000 e In the Dilution Factor box type a value from 0 001 to 1000 7 Click OK The new sample or sa
179. view The calibration curve in the Calibration Curve view opens Thermo Scientific Quantitative Analysis User Guide 59 5 Working with Calibration Settings Including or Excluding Data Points from the Calibration Curve 3 Examine the calibration curve data e To replot the data with a different X axis drag the cursor horizontally over the range that you want to expand The application rescales the axis and replots the data with the new X axis range e To replot the data with a different Y axis drag the cursor vertically over the range that you want to expand The application rescales the axis and replots the data with the new Y axis range e To cancel the replot and return to the full range of the X axis and Y axis click om 4 To exclude or include a point right click the Calibration Curve view and choose Exclusion List from the shortcut menu The Cal Exclusion List dialog box opens The application displays the level name expected amount difference and whether the point is Excluded Yes or Excluded Blank for each calibration data point on the list Excluded Blank indicates that the data point is to be included Figure 27 Cal Exclusion List dialog box Cal Exclusion List hydrocortisone Level Expected Red Sox 0 630 ng 2 029 standardl 1 250 ng 10 604 standardl 1 250 ng 2 920 standardJ 2 500 ng 4 405 Celtics 5 000 ng 0 560 Celtics 5 000 ng 0 029 standardL 12 000 ng 2077 Yes f Cancel Apply Help 5 Se
180. xes automatically For more information see Brackets and Groups for Sequences on page 12 and Bracket Group In Use List on page 91 This read only box shows the calibration method applied to the current bracket or group When the calibration information for the current bracket is obtained from the embedded processing method and not from a separate calibration file the box displays Embedded Calibration For unbracketed sequences the box displays the name of the calibration file associated with the current group in the sequence To change the named calibration file for an unbracketed sequence choose File gt Replace Calibration This option is not available for bracketed sequences 18 Quantitative Analysis User Guide Thermo Scientific 2 Quan Browser Overview Quan Browser Window Overview Results Grid View Figure 7 shows the Results grid table and shortcut menu Figure 7 Results grid table and shortcut menu Columns 4 gt All Standards QCs Blanks Unknowns Delete Selected Samples r Add Sample Copy Row Set Sorting Order Send to Qual Browser For information about the columns in this view and the shortcut menu see Quan Browser Results Grid on page 81 Chromatogram and Companion Views The Chromatogram view displays the chromatogram for the currently selected component from the currently selected result file Figure 8 shows the Chromatogram view and sho
181. y a factor of two 2 Auto Range View the chromatogram which is normalized from the minimum a eek to the maximum signal This zoom feature is recommended for PDA and UV data al Normalize Normalize the intensity scale of the data display to a fixed range on o i the Y axis for example from 0 25 to 0 100 Zoom In X To show more detail zoom in on the X axis by a factor of two 2 gt 1 y l Zoom OutX To show more detail zoom out on the X axis by a factor of two 2 from the center SS DisplayAll View all data on the X axis or all text in a report e Reset Scaling To display the maximum amount of data reset the scaling of both the X and Y axis Options Menu Table 5 Options menu commands Command Delete ComponentName Description Delete the currently selected component from analysis This command results in a recalibration Masses View or change the default settings for mass tolerance and mass precision View Stds And QCs View All Enable Warnings 76 Quantitative Analysis User Guide View either all components or just standards and quality control samples in the Results grid view View as needed all warnings boxes even though their display has been previously suppressed Thermo Scientific A Quan Browser Reference The Quan Browser Window GoTo Menu Table 6 Go To menu commands Command Description E Instrument Setup Open the Instrument Setup window a Processing Se

Xcalibur 2.1.0–2.3.0 Quantitative Analysis User Guide Version E

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Xcalibur 2.1.0&ndash;2.3.0 Quantitative Analysis User Guide Version E

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Xcalibur 2.1.0–2.3.0 Quantitative Analysis User Guide Version E