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Advanced User Guide

1. Icon Name Function Overlay Overlays graphs Cycle Overlays Cycles between overlaid graphs gt Sum Overlays Adds the graphs together m Show Fragment Opens Fragment Interpretation tool which calculates Eus Interpretation Tool the single non cyclic bond cleavage fragments from a mol file Smooth Smooths data using the smooth algorithm Gaussian smooth Smooths data using Gaussian smoothing Advanced User Guide Analyst 1 6 1 Software 57 of 94 Qualitative Data Analysis AB SCIEX Analyst 1 6 1 Software Advanced User Guide 58 of 94 Quantitative Data Analysis Calibration Options The calibration options define the parameters for a calibration curve which are used to determine the calculated concentration of the samples The curve is a plot of the concentration of the standard against the area or height of the standard if no internal standard is used If an internal standard is used the curve is a plot of the concentration ratio against the area or height ratio This curve is used along with the area or height for the unknowns to interpolate the calculated concentration Choose the best regression type or fit to fit the curve to the points and the best weighting factor for the project About Calibration Curves The calibration curve is used to determine the calculated concentration of samples including QC samples It is a curve that results from plotting the concentration of the standard v
2. Analyst 1 6 1 Software Advanced User Guide AL Release Date March 2012 This document is provided to customers who have purchased AB Sciex equipment to use in the operation of such AB Sciex equipment This document is copyright protected and any reproduction of this document or any part of this document is strictly prohibited except as AB Sciex may authorize in writing Software that may be described in this document is furnished under a license agreement It is against the law to copy modify or distribute the software on any medium except as specifically allowed in the license agreement Furthermore the license agreement may prohibit the software from being disassembled reverse engineered or decompiled for any purpose Portions of this document may make reference to other manufacturers and or their products which may contain parts whose names are registered as trademarks and or function as trademarks of their respective owners Any such use is intended only to designate those manufacturers products as supplied by AB Sciex for incorporation into its equipment and does not imply any right and or license to use or permit others to use such manufacturers and or their product names as trademarks AB Sciex makes no warranties or representations as to the fitness of this equipment for any particular purpose and assumes no responsibility or contingent liability including indirect or consequential damages for any use to w
3. In x If x lt 0 then an error is generated otherwise if x lt 10 then w In 10 otherwise w In x Use the logarithm of x to place more weight on higher value points Iny Analyst 1 6 1 Software 94 of 94 If y lt 0 then an error is generated otherwise if y lt 107 then w In 108 otherwise w In y Use the logarithm of y to place more weight on higher value points Use when calibrating by the area y axis rather than by the concentration x axis Advanced User Guide
4. 45 Display a Formula Difference in a Molecular Structure 45 IDA EXPlOrer 423i eto eoe go tace tat UR Na CHR TIME Rn RIOT eae 45 Librar Databases xo ibo nial ates ee x erbe ceti RU RA RS 46 Switch Between Existing Library Databases 00000000 47 Connect to a Local Library Database 0 000 c eee eee 48 Analyst 1 6 1 Software Advanced User Guide 4 of 94 Connect to a Server Library Database 2000000055 48 View All Library Records 22 p emen ubere redux etd ler eg cere 49 Add a Record to the Library 00 0 62 eee eee 49 Search Library Records with Constraints 00200055 50 Library Search MMPS oss steel a eae sd aa ed eae eta e ics lend en ees 51 Search for a Similar Spectrum 00 0000 cee 51 View a Compound from the Search Results 0 00000 eee 53 Processed Data Files 0 000 ee eee eee 53 Save a Processed Data File 0 0000 53 Open a Processed Data File 0 0 0 ccc eens 53 Qualitative Datas 2 521 3 ordeo teer ake i euer bed Be ere d dele Vac 53 Signal to Noise Ratio c vxo v Ed Ue pM Xm EIE MESA EE 54 smoothing Algorithm S ease ai esed a x PR eR eh eae D e E e 54 Smooth Data using the Smooth Algorithm 0c cee ee eee 55 Smooth Data using Gaussian Smoothing 0 00 e eee ee eee 55 Spectral Arithmetic Wizard 0 0 0 ccc eee ees 56 Toolbar Icons 3x das Preces
5. n n anaa aaan 70 Confirm the Peak Start llli 70 Find the Peak Top su ese ee nia Oa RR CR S Re OR es Grade 71 Find the Peak End exei ket kr EE UNE rE EH EE RERCR P UR Wh 73 Separate Peaks stre roe e e PRESENT RAIN ae Sa aa dfe 74 Queres eraren RAE Ane afe 4 by wire ee PAD AY Hig bod AG Rik ey OE Chen 75 Queries on Sample Type 25 e eee ee eee ums 75 Default Queries and Table Specific Queries 0020005 75 How Accuracy Variations Affect the Results 0500055 76 FREQLOS SION sexes Tc Gan Bys das aa ween dens he Aid ay oS Such Raed 76 Advanced User Guide Analyst 1 6 1 Software 5 of 94 Linear Regression e Loses mre tM I EA MIEL EUM mE dU oL tM d 77 Linear Through Zero Regression 5 0002 eee eee 77 Mean Response Factor 000 000 eee 77 Power soe ades Nuus ef eet e a ane be dae ye PN EP S RO ah 78 Quadratic Calibration Equation 202 0c 78 Report Templates 0 00 c cette 79 Customize Reports 0 0 0 cece ns 81 Preview Print and Export Reports 0 0 ccc eee eee 81 Results Tables i e reat be ae es DI ul A qo Lal esa ee dod 82 Define the Layout of Results Tables 20200000000005 82 Sort Data in Results Tables 0 0 2 0 0 0000 0c eee 83 Sort a Results Table and Save the Sort Criteria 83 Save Default Sort Criteria for Future Results Tables 84
6. the peak The software displays the Peak Review window with the user selected peak Results Tables To return the Right click on the Results Table and click Sort Sort By Index Results Table to its original order Weighting Factors The following table shows how the weighting factor w in the equations below is calculated for each of the seven weighting types Refer to Regression on page 76 Table 6 19 Weighting Factors Weighting Type None Weight w Always 1 0 1 x If x lt 107 then w 10 otherwise w 1 x To place some additional emphasis on lower value points use a weighting factor of 1 x 1 x2 If x lt 107 then w 10 otherwise w 1 x Use a weighting of 1 x squared to provide a much higher emphasis on lower value points 1 y If y 1078 then w 10 otherwise w 1 ly Use a weighting factor of 1 y when calibrating by the area y axis rather than by the concentration x axis and some emphasis needs to be placed on lower value points A weighting of 1 y is a variant of 1 x where y and x should be proportional to each other 11y If y 107 then w 1076 otherwise w 1 y Use a weighting factor of 1 y squared when calibrating by the area y axis rather than by the concentration x axis and extra emphasis needs to be placed on lower value points A weighting of 1 y is a variant of 1 x where y and x should be proportional to each other
7. 1367 3948 0083 ER 391 5188 0083 ER 394 3800 0328 ER 387 5343 0369 ER 224 i 1 00 4 20e8 i 0 804 0 604 a 1395 4684 0410 ER 445 1524 0410 ER z 416 9524 1140 ER B 9 429 0503 1 687 ER i 3732604 2167 ER 0 204 L 4753677 2562 EP 16 U 357 3078 2736 EP 11 U PEN 1 475 3800 2 537 ER 5 10 15 2n 25 35 40 45 0 5 L 4124081 2628 ER mz Da C 440 3400 2 628 ER EE XIC of EMS Exp Max 2 2e5 ops E Max 1 0 c L 4450434 2669 ER L 3573866 2710 ER 3 2e5 a 1 004 1566 0856 2710 ER 3 0054 359 2365 2777 ER i4 oso 1566 2103 2777 ER 2 5e5 0566 2131 2802 EP 20 U amp sons S l 36726982 2843 ER E i E 5002743 2843 ER Z 1 5054 B 1500 2775 2868 EPI 38 1 2 i oe L 3514388 2909 ER m aos D L 3631800 2950 ER j 0 204 383 3072 307 EP 12 uU x seti 0 0 Pi E C Tree View List View 5 10 15 20 25 30 35 40 45 05 0 Figure 5 4 IDA Viewer Library Databases The Library Search feature compares unknown spectra to known MS spectra contained in a library database and generates a list of possible matches Use Library Search to create and manage a mass spectra database that can be used to search for and match unknown spectra against the mass spectra stored in the database With Library Search users can Compare library contents against an unknown spectrum Add records to the library Edit existing records Library data can be
8. Ethanol 114 Ethyl acetate 104 Heptane 120 Hexane 150 Isobutanol 100 Isopropanol 100 Methanol 120 1 Propanol 100 Toluene 87 Water 46 Syringe Size Versus Flow Rate The flow rate of a syringe pump depends on the syringe installed in the pump The following tables show the relationship between flow rate and syringe size Advanced User Guide Analyst 1 6 1 Software 29 of 94 Device Methods Table 4 2 Syringe Size and Flow Rate at L hour Syringe size L hour pL Minimum Maximum 0 5 002 23 8 1 0 003 47 8 2 0 006 95 2 5 0 015 238 0 10 0 029 474 0 25 0 073 1193 0 Table 4 3 Syringe Size and Flow Rate at uL minute Syringe size pL minute pL Minimum Maximum 50 002 39 7 100 005 79 7 250 012 197 8 500 024 397 0 1000 048 795 0 1 0 049 805 0 Table 4 4 Syringe Size and Flow Rate at mL hour Syringe size mL hour mL Minimum Maximum 2 0 011 186 8 2 5 010 168 2 3 0 011 181 4 5 0 019 317 0 10 0 028 461 0 20 0 050 821 0 30 0 074 1208 0 Table 4 5 Syringe Size and Flow Rate at mL minute Analyst 1 6 1 Software 30 of 94 Syringe size mL minute mL Minimum Maximum 50 0 002 28 40 100 0 003 47 60 140 0 004 55 10 Advanced User Guide Qualitative Data Analysis Users can view the information contained in a data file in table or graph form Graphical data
9. e Oo Hi B series 2 0 ng mL GuanDats Wit 1 50e 004 477e 003 0 00 m mj M3 E series 5 0 ng mL QuanData Witt 3 70e 004 1 206004 0 00 v mr 5 B senes 10 0 ngm QuanDats Wit 7 73e 004 2 488 004 0 00 m D B senes 20 D ngfmL QuanData Witt 7 51e 004 2 44e 004 0 00 e Oo 119 Unknown concentra QuanData Wit 1 23e 004 4 30e4003 WA r 21 Unknown concentra QuanData Wif 8 71e 003 2 5394005 WA m 23 Unknown concentra QuanDats Wiif 1 12e 004 3 40e 003 WA m 25 Unknown concentra QuanDats Wit 1 32e 004 4 24e 003 WA D Unknown concentra GuanDats Wii 1 25e 004 4 04e 003 WA r 29 Unknown concentra QuanData wii 1 108204 3 960 003 NA Hn 31 Unknown concentra QuanData Wi 1 36e 004 5 16e 03 WA n Figure 6 17 Sample Analyte Layout view Advanced User Guide Analyst 1 6 1 Software 87 of 94 Quantitative Data Analysis Analyte Group Layout View The Analyte Group Layout view contains the data for the analytes that belong to a particular group Columns that are selected as shown in the Results Table Columns dialog appear in the Results Table as shown in Figure 6 18 Display the Analyte Peak Name column in the Results Table to show the names of the analytes that belong in the group Formula Analyte Group Minoxidds Only i 2 i DA Query None Idle Sort Unsorted Analyte Peak Em Sample Name Sample ID Sample Type Name It STD 1 Standard Mix batch 1 wif minoxidol 2 sTD 1 Standard Mix batch 1 wiff minoxidol STD 2 Standard
10. obtain as much information as possible in the same run Figure 4 2 shows a three period method The first period has a duration of 3 015 minutes the second period is 4 986 minutes and the third period is 7 000 minutes Advanced User Guide Analyst 1 6 1 Software 27 of 94 Device Methods Acquistion methed MS Advanced us r C B Acquistion Method c n a Center With 49 Mass spec 24 003 min v Parameter Rance Picea St T9 n m Scantype Product lon MSD E mu eB 4s2 amp Penod 5 025 mn 2 4 Agent 1100 LC Binary Pump 0 0 mns ec Equilbrate 0 0 mns 3 Run 0 0 mine Product OF 582 Do Agilent 1100 Colum Oven Agilent 1100 Autosampier Tota Scan Time n includes paueec S qe Period Summary Edt Paremetes Duration 13570 mn Delay Time O osc Cycles 47 Cycle 03 eo Figure 4 2 Example of a multi period experiment Information Dependent Acquisition Methods Information Dependent Acquisition IDA is an acquisition method that analyzes data during acquisition IDA is used to change the experimental conditions depending on the analysis results These real time changes are controlled by criteria set in the acquisition method including e lon intensity and charge state Inclusion and exclusion lists e Isotope pattern Optimizing data acquisition settings while the data is being acquired allows users to conserve both the sample an
11. the value of that threshold at 0 000001 Peak Integration The following are integration types by which the baseline was found and integrated when the peak was found e Manual The peak was manually integrated by the user Automatic The peak was automatically integrated as follows Baseline to baseline The peak area is defined by vertical droplines at the beginning and end of the peak which extend to the baseline This integration type is possible only for peaks that do not have another peak immediately preceding or following Valley Same as baseline to baseline except that it applies only to peaks that do have another peak immediately preceding or following Exponential Skim The peak area is the main or parent peak in an exponential skim Exponential Child The peak area is the child peak resulting in an exponential skim Peak Review During peak review users can survey the peaks that the software selected and then redefine the peak or the start and end points where necessary In general the software is adept at accurately identifying analyte and internal standard peaks For a variety of reasons including sample acquisition and quantitation method definition sometimes the software misses the correct peak chooses the wrong one or is unable to locate a Analyst 1 6 1 Software Advanced User Guide 68 of 94 Quantitative Data Analysis peak at all Other times although the software may correctly identify th
12. to the lonSpray ion source voltage Period and Experiment A period contains a collection of experiments an experiment contains a number of properties such as Scan Type Scan Mode Resolution lon Source Parameters and a collection of mass ranges or masses State Table Parameters The instrument parameters used in the experiment Pump The name of the pump used for the experiment Autosampler The name of the autosampler used for the experiment Custom Annotation The custom text added via the Batch Editor Collected By The name of the person who collected the data Table 6 10 Quantitation Elements Element Results Table Name Definition The name of the Results Table Results Table Path The location of the Results Table Method Name The name of the quantitation method Method Path The location of the method file Project Name The name of the project Results Table Name Analyst 1 6 1 Software 80 of 94 The name of the Results Table Advanced User Guide Quantitative Data Analysis Customize Reports The Report Template Editor provides a way to customize reports by setting up headers footers and page layouts Use report templates with both printed output and data exported to another application Printed output includes several types of elements Window Windows in the Analyst software appear in the working area of the soft
13. 008 2 23e 003 0 00 B B series 1 0 ngmL Siandard QuanDats Witi Feak 2 1 23e 004 4 e03 0 00 M B series 20 nm Standard QuanDats Wit Peak 1 1 5De 004 477e 003 1 00 B series 2 0 ng mL Standard QuanData Wit Peak 2 1 34e4004 4 3e4003 ntn B serias 5 0 ngmL Standard QuanData Wit Peak 1 3706 004 1206 004 0 00 B series 50 ng mL Siandard QuanDats Wil Peak 2 1 51 6 004 5 Z8e 2C3 0 00 B seres 100 ngm Standard QuanData Wit Peak 1 7 T3e 004 2 49e 004 0 pa MB B series 10 0 ng mi Stsndsrs QuanDats Wit Peak 2 1 50e 004 5 41e 003 n en E seris 20 0 ng mStandard GuanData Wf Peak 1 7 Bie I04 24404004 0 00 18 B senes 20 0 ngfmUStandsrd QuanDats Will Peak 2 B 04e4203 3 13e 203 0 00 Advanced User Guide Quantitative Data Analysis Summary Layout View The Summary Layout view contains the locked columns and the chosen field for each analyte in the remaining columns For example if Analyte Peak Area is selected from the menu for two analytes then the Sample Name and Analyte Peak Area columns for those analyte names are seen The Summary Layout view also includes the Formula and Custom columns if these exist B seresQ blank 2 45e 002 12504204 B senes 0 1 ngfmL 7 80e 002 1 28e 004 B senes 0 2 ng mL 1 55e3003 1 zBe 0D4 B series 05 ng mL 3 32e 003 1 14e 004 I E series 1 0 ng mL 7 12e 003 1 2304004 11 B senes 2 0 ng mL 1 50e 004 1 348004 Em B
14. 1 describes the contents of the different folders The software can access a project only if it is stored in a root folder Users cannot create projects in a folder that has not been defined as a root folder The preset root folder is Analyst Data on the drive where the software is installed To store projects in other locations create new root folders For more information about root folders refer to the Help Table 1 1 Project Folders Folder Contents Acquisition Methods Contains all acquisition methods used Acquisition methods have the dam extension Acquisition Scripts Contains all the acquisition batch scripts available Batch Contains all the acquisition batch files used Acquisition batches have the dab extension It also contains a subfolder Templates that contains acquisition batch templates Batch templates have the dat extension Analyst 1 6 1 Software Advanced User Guide 12 of 94 General Information Table 1 1 Project Folders Continued Folder Contents BioAnalyst Contains files used with the BioAnalyst software add on a protein analysis tool The folder is available only when the BioAnalyst software is installed Data Contains the acquisition data files wiff extension Log Contains results of quantitation and compound optimization Processing Methods Contains all qualitative data processing methods used Processing Scripts Contains all data processi
15. Search Constraints dialog 2 In the Maximum Number of Match field type the maximum number of compounds to be returned by the search 3 In the Preselect Constraints section select the check boxes for the constraints to apply For each constraint selected in the Preset Tolerance section type the tolerance If required select a method of sorting records from the Result Sorted by list If required type text in the Comment Contains field If required type text in the Keyword Contains field pool m ou 4 To apply peak constraints by adding and removing peaks click Peak Constraints The Peaks Included table opens 9 To add peaks to the list to search against click Add and then type the m z and the corresponding intensity in the empty cell 10 To remove peaks so that they will not be included in the search select the peaks and then click Remove 11 Click Search to save the constraints and begin the search Analyst 1 6 1 Software Advanced User Guide 52 of 94 Qualitative Data Analysis View a Compound from the Search Results If several spectra match the unknown spectrum the user may want to view other spectra and compare them to the unknown 1 In the Search Results dialog in the list of compounds select the row number of the compound 2 Click the spectrum pane of one of the known compounds The spectrum of the selected compound is shown Processed Data Files The user can save processed data such as specifi
16. TIC XIC TWC or XWC graph 2 Highlight the range to be viewed in the Contour Plot If a selection is not made the entire range is viewed 3 Click Explore Show Show Contour Plot A Contour Plot of the selected area opens in a separate pane Select an Area in a Contour Plot To zoom in on a particular selection or view the corresponding mass spectrum for that selection do one of the following Advanced User Guide Analyst 1 6 1 Software 41 of 94 Qualitative Data Analysis To select a standard area within a box drag the pointer to create a box around an area in the Contour Plot To make a vertical selection press Ctrl and drag the pointer vertically To make a horizontal selection press the space bar and drag the pointer horizontally Set the Intensity and Absorbance in a Contour Plot Do one of the following To set the low intensity absorbance value in a Contour Plot from the color bar above the Contour Plot drag the left triangular slider to the required position Contour Plot automatically adjusts the color of values below the setting to indicate they are outside the range To set the high intensity absorbance value in a Contour Plot from the color bar above the Contour Plot drag the right triangular slider to the required position The Contour Plot automatically adjusts the color of values above the setting to indicate they are outside the range Change Colors in a Contour Plot
17. Table 6 4 shows the parameters available with the MQ III algorithm but not the IQA II algorithm Table 6 4 MO III Algorithm Parameter Default Noise Percentage Definition The threshold used in peak finding Only peaks higher than this specified percentage will be detected Default Baseline Subtraction Window A time window around each data point that is used to determine the height of the baseline correction to be applied to that point This time window helps remove excessive noise from the chromatogram The baseline is defined as the line connecting the point of minimum intensity on the left side of a given data point to the point of minimum intensity on the right side within the specified window Default Peak Splitting Factor Controls whether a given peak cluster consists of multiple adjacent peaks or one possibly noisy peak If the intensity dip is less than the value specified then a single peak is reported otherwise the point with minimum intensity in the dip splits the cluster into two separate peaks Setting a large factor will prevent clusters from being split into more than one peak Default Void Volume Retention Time Advanced User Guide Any peaks that appear before this time are ignored Analyst 1 6 1 Software 61 of 94 Quantitative Data Analysis Table 6 4 MO III Algorithm Continued Parameter Definition Report Largest Peak Selecting this parameter returns the largest peak in
18. The name of the data file with the sample acquisition information Acquisition Date The date the sample acquisition was run Acquisition Time The time of the sample acquisition was run Operator The name of the operator who ran the sample batch Advanced User Guide Analyst 1 6 1 Software 79 of 94 Quantitative Data Analysis Table 6 9 Acquisition Elements Continued Element Batch Name Definition The name of the batch Sample Number The number related to the sample Sample Name The name of the sample Sample Comment Shows any comments about the sample entered through the Acquisition Method Editor Sample ID The identification number of the sample Scan Mode Indicates the method in which the system calculates the mass points for a scan for a full mass range scan Scan Type and Polarity Indicates the acquisition scan type Q1 Q3 MRM Product ion Precursor ion neutral loss gain and acquisition method polarity positive or negative Scan Mass es lons or ion fragments to be scanned Dwell Time The time the system takes to scan a particular mass Pause Time Indicates the pause between the scanning of mass ranges or between experiments lon Energy lon energy comes from the acquisition method and is related to the lonSpray ion source voltage or the collision energy Collision Energy Collision energy comes from the acquisition method and is related
19. calculate the noise the software uses the standard deviation using a mean of zero of all data points in the chromatogram from the Background Start to Background End time both shown in the advanced parameters for the Quantitation Method Editor and Peak Review window These times are set when a new background range is defined If the user builds a method without defining a new background range which is possible if the preset integration is accepted with no changes then the value for both the Background Start and the Background End is shown as N A As a result the signal to noise ratio is not calculated and the corresponding field in the Results Table is shown as N A Smoothing Algorithms The user can select either the smoothing algorithm or the Gaussian smoothing algorithm as the smoothing method The smoothing operation involves replacing each data point with the average of the data point before and after it The smoothed data set replaces the old set Data can be smoothed more than once but the software can undo only the last smooth Smoothing is not available for multiple ion or MRM spectra Smooth Algorithm When smoothing data the user sets the point weighting values for three data points the current point the preceding point and the following data point The smooth algorithm multiplies the data points by the assigned weighting values sums these values and then divides the total by the sum of the point weight values It is a gen
20. e CSV doc e pdf e txt The formats available depend on the information being exported For example a graph can be exported as a pdf a table of data can be exported as a txt file To include additional information in the header and footer of the report print the report using an appropriate template Table 6 11 Previewing Printing and Exporting Reports To do this do this Preview a graph 1 Click File Print Preview Pane 2 Edit the Print Preview dialog 3 Click Print Advanced User Guide Analyst 1 6 1 Software 81 of 94 Quantitative Data Analysis Table 6 11 Previewing Printing and Exporting Reports Continued To do this do this Print a report without a Click File Print and then click the report template Print a report with a template 1 Click File Print amp Report Setup 2 In the Report Template section select the template 3 Click OK Export a report 1 Click File Export 2 In the File field type the name of the file 3 In the Save as type list select the appropriate file type 4 If exporting a report in Quantitate mode select either All Columns or Visible Columns from the Export section and then click Save Results Tables Results Tables summarize the calculated concentration of analyte in each unknown sample based on the calibration curve They also include the calibration curves and statistics for the results Export the data from a Re
21. from the spectrum 2 In the Mass Spectral Information tab type a name in the Compound Name field The compound name is mandatory and must uniquely identify the compound within the library 3 Edit any of the other fields Many of the fields are filled in automatically from the data associated with the spectrum Click the General Information tab Edit the fields as required Click OK Search Library Records with Constraints 1 Click Explore gt Library Search gt List With Constraints List Constraints m Conditions Field Name Relation Value v Contains E Elements Included Excluded Cancel List Figure 5 8 List Constraints dialog In the Field Name list select a field on which to base a constraint In the Relation list select the relation operator that applies to the field name In the Value field type the value of the field name based on the relation To add the selected constraint to the Conditions list click Add Continue to add constraints to the conditions list as required DUOIDE OUR di du NS Coupling distinct constraints within the Conditions list creates more specific conditions that enhance the search To group constraints select the constraints and then click Group To separate grouped constraints click the group and then click Ungroup Analyst 1 6 1 Software Advanced User Guide 50 of 94 Qualitative Data Analysis 8 Tochange the relationship between constraints clic
22. i e e om Ae e OR de SOROR ID aR Re es 56 Chapter 6 Quantitative Data Analysis lees 59 Galibration Options s o Puoi ew y vesc eye i que RYE E Fa 59 About Calibration Curves llli eee 59 Select the Best Regression Type 020 000 e eee eee eee 59 Integration Algorithms ec kp eid dvs Rex eke ee KE Aud 60 Analyst Classic and IntelliQuan Integration Algorithms 61 Quantitation Method Creation Tools 2 00002 ee eee eee 62 Wizards 25 crean witty C EE oe cte ie ant Rogo ated ye te cee E Ae 62 Find Peaks Using an Automatic Method 2000200005 63 Quantitation Method Editor l l 63 The Semi Automatic Method Editor 00 00sec eee eee ees 63 Find Peaks Using a Semi Automatic Method 2 2 64 Metric Plots so Rex e ath alae Dee Rag eed ed Ltd d d 64 Generate a Metric Temporary Plot lille 65 Generate a Metric Plot and Save the Plot Criteria 65 Save Default Plot Criteria for Future Results Tables 67 Noise and Area Threshold Parameters llli lesen 67 Recalculate the Noise and Area Threshold 2 020200055 68 P ak Int gration MCCC EE 68 Peak REVIEW c sore nx nep iR EO IU ue REO SOE HEROS RUMOR eos Debra Poe e 68 Peak Review TIDS xs eee Rue ue ce eR ERATES REA 69 Detect Peaks 4 exit ele a ei ERR NUR ERES e NE 69 Find the Potential Peak Start
23. masses from single non cyclic bond cleavage of a molecular structure The molecular structure can be created in a third party drawing program and then saved as a mol file Fragment Interpretation displays the theoretical fragments in the fragment list and compares the fragment masses to peaks in the mass spectrum Peaks above the threshold intensity and within the user defined mass tolerance maximum 2 amu of fragment masses are considered matched and appear in bold text in the fragment list 7 Note The Fragment Interpretation tool cannot be used with the following scan types e Precursor lon e Neutral Loss e Q1 Multiple lon Q3 Multiple lon Multiple Reaction Monitoring MRM Connect the Fragment Interpretation Tool to a Spectrum When a single non cyclic bond in the molecular structure is selected the Fragment Interpretation tool highlights the two fragments created when the bond is cleaved and matching peaks in the connected spectrum are displayed If multiple spectrum panes are being viewed then the Fragment Interpretation tool connects to the active spectrum If the data file contains more than one sample then the Fragment Interpretation tool connects to the active spectrum If a spectrum is open when the Fragment Interpretation tool is opened then the active panel links to the open spectrum automatically 1 Click Explore Show Show Fragment Interpretation Tool 2 From the lower right corner of the Fragment In
24. message box Analyst 1 6 1 Software Advanced User Guide Qualitative Data Analysis Display a Formula Difference in the Fragment List 1 Click the row number for one fragment 2 Press the Ctrl key and then click another fragment The formula and monoisotopic mass difference is shown in a message box if the fragments are related Display a Formula Difference in a Molecular Structure 1 Click a single non cyclic bond The preset fragment of the two highlighted fragments is selected To select the other fragment of the cleaved bond CTRL click the bond 2 Select a second non cyclic bond To select the preset fragment press the Shift key and then click the bond To select the other fragment of the cleaved bond press Ctrl Shift click the bond Fragment Interpretation calculates the formula and monoisotopic mass difference between the fragment selected in step 1 and the fragment selected in step 2 if the fragments are related The formula and monoisotopic mass difference is shown in a message box IDA Explorer The Information Dependent Acquisition IDA Explorer is used to display data acquired through an IDA method The IDA Explorer can be turned off and on in the IDA Explorer tab in the Appearance Options dialog Columns present in the List View can be defined in this tab as well The left side of the viewer shown in Figure 5 4 displays the masses on which a product ion scan was performed In this area users can examine th
25. of 94 Quantitative Data Analysis Find the Potential Peak Start To find the potential start of a peak the software measures the intensity difference between sequential pairs of bunched points starting at the first point When it finds a difference that exceeds the current noise threshold the software declares the first point a potential peak start oN o Figure 6 3 Finding the potential peak start Item Description 1 Exceeds noise threshold 2 Potential peak start 3 Does not exceed noise threshold Confirm the Peak Start To make sure that it has found a real peak the software moves along the curve adding the intensity difference between each bunched data point from the intensity at the potential peak start to a total sum This process stops when the intensity difference between successive points is less than the noise threshold This sum is an approximation of the area of the leading edge of the peak If this sum exceeds the area threshold then the software confirms the peak start Next the software determines the actual start of the peak by moving backward from the potential peak start until it finds the lowest point in the peak It moves back through five bunches of raw data This point is the actual peak start Analyst 1 6 1 Software Advanced User Guide 70 of 94 Quantitative Data Analysis v Figure 6 4 Confirming the peak start Item Description 1 Su
26. open in the Acquisition Method Editor in the Acquisition method pane click the Pump icon The Pump Properties tab opens in the Acquisition Method Editor pane Edit the fields as required 3 Save the file Set the Autosampler Properties 1 Make sure that on the Acquisition Properties tab the Synchronization Mode field is set to LC Sync The device and the instrument will start simultaneously 2 With a method file open in the Acquisition Method Editor in the Acquisition method pane click the Autosampler icon The Autosampler Properties tab opens in the Acquisition Method Editor pane 3 Editthe fields as required Save the file Set the Column Oven Properties 1 With a method file open in the Acquisition Method Editor in the Acquisition method pane click the Column Oven icon The Column Oven properties tab opens in the Acquisition Method Editor pane 2 Editthe fields as required Analyst 1 6 1 Software Advanced User Guide 24 of 94 Device Methods 3 Save the file Set the Switching Valve Properties The switching valve can be used as a diverter or injection valve Select the Manual Sync with Valve synchronization mode if using the valve as an injector choose any other mode if using the valve as a diverter 1 With a method file open in the Acquisition Method Editor in the Acquisition method pane click the Valve icon The Valve Properties tab opens in the Acquisition Method Editor pane 2 Change the positio
27. peak area or height The exact variables used for the regression depend on whether or not an internal standard is being used and whether the peak area or the peak height is used as shown in the following table Table 6 7 Regression Formulae Internal Standard Used J Area Used x y Yes Yes C C DF Al Ais Yes No C C DF H His No Yes C DF Ag No No C DF Ha Where e C actual analyte concentration e Cj internal standard concentration Analyst 1 6 1 Software Advanced User Guide 76 of 94 Quantitative Data Analysis e DF dilution factor e A analyte peak area Aj internal standard peak area H analyte peak height e Hi internal standard peak height Linear Regression Linear regression assumes that the points of the standard fall on a straight line The linear calibration equation is calculated as y mxt b The slope and intercept are calculated as m ZwX wxy X wxX wy Dx b Ewx2Xwy XwxX wxy Dx The correlation co efficient is calculated as r ZwXwxy ZwxZEwy A DxDy where Dx EwXwx2 Xwx 2 Dy XwXEwy Xwy Linear Through Zero Regression Linear through zero regression assumes that the points of the standard fall on a straight line and that the points do line up with the zero point on the x and y axes Use this setting to force the line to go through the zero point The linear through zero calibration equation is calculated as y mx The s
28. peak review as users can see the peaks with low Analyte Integration Quality values for manual review In addition users can query the data for the Analyte Integration Quality values that are less than a value they consider acceptable in order to display and manually review a subset of the data The Sample Column fields display information about the sample that is common to all analytes Blank and double blank can be defined differently from lab to lab Table 6 15 shows the available fields Table 6 15 Sample Column Fields Field Sample Name Definition The name that the user assigned to the sample when it was acquired Sample ID A user defined identifier associated with the sample Sample Type All analytes within a sample must have the same sample type One of the following sample types is displayed Unknown Contains analytes whose concentrations are to be determined Standard A sample with known analyte concentration used for calibration purposes Quality Control A sample with known analyte concentration used to check the accuracy of the standard curve Solvent Confirms that the instrument is clean Solvents are not run through the sample preparation process Blank A zero concentration sample that is not used in regression Double Blank A sample prepared without an internal standard or sample analyte confirms that the extraction process added nothing Sample Comment A comment descr
29. sample details such as the sample name and the acquisition method to be used to acquire the sample Locations Used to select the positions of samples in the autosampler Sample locations can be specified numerically in the Sample tab however the Locations tab provides a graphical interface for selecting sample locations Quantitation Used to select the sample types and concentrations for quantitation batches Because quantitation information can be specified post acquisition in the quantitation Results Table users do not have to use the Quantitation tab in the Batch Editor Instead the Quantitation Wizard can be used Advanced User Guide Analyst 1 6 1 Software leas March 2012 19 of 94 Batches Table 3 1 Batch Editor Tabs Continued Tab Description Used to verify sample information and to submit samples to the acquisition queue The Queue Manager shows queue batch and sample status and allows users to manage samples in the queue Batch Files To make data entry easier batch file information from other applications can be imported The Batch Editor can import text and other files that are formatted correctly For examples of correctly formatted files refer to the Batch folder in the Example project The information in a batch file can also be exported for use with other applications such as Microsoft Excel Microsoft Access and certain LIMS Laboratory Information Management System software Bui
30. stays in the same place relative to the x and y axes when the graph is zoomed in or out To edit or delete the caption right click the caption and then click the appropriate command Advanced User Guide Analyst 1 6 1 Software 39 of 94 Qualitative Data Analysis Add Text to a Graph Use text to add multiple lines of information to a graph Unlike captions which are associated with a specific peak and move with it as the graph is zoomed text labels remain in their original location as the graph is zoomed They do not stay with the original sample when users navigate between samples in a data file 1 Inthe graph right click and then click Add User Text The Add User Text dialog opens In the User Text field type the text To center the text select the Center Text check box To change the size and style of the caption click Font To insert the text click OK Bode OO o different position To edit or delete the text right click the text and then e Tip Ifthe position of the text is not satisfactory then drag the text to a choose the appropriate command Compound Database The compound database stores information about compounds including optimization specifications Use the compound database when there is a large numbers of samples and a large number of compounds need to be optimized quickly The Compound Database window stores optimized conditions for compounds that can be retrieved to run samples
31. the retention time window If this parameter is not selected the closest peak to the expected retention time is found The expected retention time is automatically calculated in the Quantitation Wizard Table 6 5 shows the parameters available for use with both IntelliQuan algorithms Table 6 5 IntelliQuan Algorithm Parameter Definition Default Minimum Peak Height The minimum height of a peak required for peak integration Default Minimum Peak Width The minimum width of a peak required for peak integration Default RT Window Specifies the time window centered at the expected retention time for peak finding For example a 30 second retention time window gives an additional 15 seconds before and after the expected retention time Default Smoothing Width The number of points used in data smoothing Default Concentration Units The concentration units used to describe the sample concentration for example pg uL Default Calculated Concentration The concentration units used to describe the calculated Units sample concentration for example pg uL Quantitation Method Creation Tools The software offers four quantitation method creation tools each of which creates a fully functional method The best choice of tool depends on the tasks to accomplish Wizards There are two available method creation wizards the Standard Quantitation wizard and the Automatic Quantitation wizard Both al
32. the internal standard is given IS Peak Area The area of the internal standard peak IS Peak Height The height of the internal standard peak Analyst 1 6 1 Software Advanced User Guide 88 of 94 Quantitative Data Analysis Table 6 12 Internal Standards Column Fields Continued Field IS Concentration Definition The known concentration of the internal standard this applies to standard and quality control sample types Zeroes are displayed for solvent blank and double blank sample types N A is displayed for unknowns IS Retention Time The chromatographic retention time as determined by the software IS Expected Retention Time The retention time of the representative sample Taken from the quantitation method IS Retention Time Window The retention time window as specified in the quantitation method IS Centroid Location The intensity weighted average retention time for the analyte The peak areas up to and after this time are identified IS Start Scan The cycle number associated with the period experiment combination where the peak begins IS Start Time The time associated with the period experiment combination where the peak begins IS Stop Scan The cycle number associated with the period experiment combination where the peak ends IS Stop Time The time associated with the period experiment combination where the peak ends IS Integration Type The method by which the
33. 00 eee eee eee 16 Compound Optimization 00 000 16 Flow Injection Analysis ur y aaua TE GO RR Races ewes ee RR ede a 16 Inih MM TDET 17 Chapter 3 Batches scocc e e n a ra oma ya asser REEEC 19 Balch Editor asus Ea xe pr erc ERE TRE ERE SAX FEN E va e Y 19 Batch Files saie aana aE araa R E Ra a Aa E e AR A E rrr 20 Build a Batch as a Text File llle 20 Import a Batch asa Text File lille 20 Set Quantitation Details in the Batch Editor Optional 21 Chapter 4 Device Methods lslleeleeeeeeleeeeee 23 Devices in Acquisition Methods 2000 e eee 23 Add or Remove an LC Device 0 0 eee nas 23 Set the LC Pump Properties llli 24 Set the Autosampler Properties 0 000 cece ees 24 Set the Column Oven Properties 00 0c cee eee eee eens 24 Set the Switching Valve Properties llle 25 Set the Diode Array Detector Parameters 0 000 cee eee eee 25 Set the Analog to Digital Converter Properties 00000005 25 Dynamic Fill Time rrr rm 27 Experiments and Periods 0000 ccc eee eee 27 Experiments xs xac ee dak Rae RECON ER CIAR Rh ce Re C ee A 27 PElOGS iid ehh co eed DA he es be ee Me es a a 27 Information Dependent Acquisition Methods llle 28 Solvent Compressibility Values lille 29 Syringe Siz
34. Analysis e To view the Analyte layout click Analyte and then click a single analyte if more than one analyte exists To view the Analyte Group layout click Analyte Group and then click an analyte group Tip Anew analyte group must be created first To do this right click in the Results Table and then click Analyte Group New b d The table opens with the selected layout e Tip To go back to the full view right click and click Full i gt Sort Data in Results Tables 1 Select up to three columns in the Results Table in the order they need to be sorted 2 Doone ofthe following To sort in ascending order click A Z To sort in descending order click Z A Sort a Results Table and Save the Sort Criteria 1 Right click in the Results Table and then click Sort New Sort By Group ft Ascending Column v Descending ThenBy Group Off Ascending Column Descending Then By Group Off Ascending Column v O Descending Figure 6 12 Sort dialog 2 In the Name field type the name for the new sort Advanced User Guide Analyst 1 6 1 Software 83 of 94 Quantitative Data Analysis 3 For each sorting rule to be set in the Sort By section do the following i Inthe Group list select the type of column ii In the Column list select the column iii Select the direction of the sort Ascending or Descendin
35. Contour Plots A Contour Plot is a color coded plot of a complete data set that uses color to represent a third dimension in the plot In a Contour Plot of a TIC the x axis represents retention time or scan number the y axis represents mass and the color represents the intensity of the data at that point In a Contour Plot of a TWC for DAD data the x axis represents retention time or scan number the y axis represents wavelength and the color represents absorbance The Contour Plot is a post acquisition tool that does not function in a real time scan acquisition Scans Jl Note The Contour Plot does not support MI or MRM scans but it does support DAD Color is the third axis in Contour Plot and it represents either intensity or absorbance Users can change the high and low intensity or absorbance values in Contour Plot using the control triangles on the color bar above the Contour Plot The percentage parameters at the top of the Contour Plot pane indicate the values held by the low and high sliders The actual values are based on a percentage of the maximum intensity or absorbance within the selected area The value is shown in the top right corner of the Contour Plot pane The controls shown in Figure 5 3 change the colors in a Contour Plot Analyst 1 6 1 Software Advanced User Guide 40 of 94 Qualitative Data Analysis No Data Low Data High Data 0 0 100 0 a a Below Low Data Above High Data Figure 5 3 Buttons Contr
36. Mix batch wif minoxidol STD 2 Standard Mix batch 1 wif minoxidol STD 3 Standard Mix _batch_1 wiff minoxidol ee IST 3 andard Mix bo xf Tie Figure 6 18 Sample Analyte Group Layout view Results Table Fields Add columns to the standard Results Table to display DAD diode array detector data for the Analyte Internal Standard and Record fields Formula Fields The Formula fields display the result of a spreadsheet style formula defined by users The Formula field located at the top of the Results Table is shown only if at least one Formula column is in the Results Table The Formula field becomes active when Formula column cells are selected The Delete Formula Column button below the Formula field also becomes available when the Formula column is selected Custom Fields Custom fields contain information defined during the acquisition process When acquiring samples users can create custom columns and define the type of data that goes in them Once the custom column is part of the Results Table it can be treated like any other column for example move it hide it base a formula on it Internal Standards Column Fields The Internal Standard Results fields display information about the internal standard after analysis Table 6 12 shows the available fields Table 6 12 Internal Standards Column Fields Field Definition IS Peak Name The name of the internal standard peak IS Units The units in which
37. Sort a Results Table using Preset Sort Criteria 85 About using Queries with Results Tables 00020 eee 85 Compare Results Between Batches 0 00 cece eens 85 How Concentration Levels Affect Results 000 0 ee eeee 86 Results Table Layouts came oe ang eder SRM UR a ee en aoe UR 86 Summary Layout View exe IIR he ee Ok new eR doe eed Ree eRe 87 Arialvie LAVOUL MIB actu 2 93 52 oid Sue Sade rv eel apum P 4 44 87 Analyte Group Layout View slsieeeeeeee rhe 88 Results Table Fields illii 88 Results Table Tips lllsllelle sh 94 Weighting FaCtOrS s eor onem wand tte CRUS Tir earth hen Saeed BRE RRR 94 Analyst 1 6 1 Software Advanced User Guide 6 of 94 Foreword This Advanced User Guide provides information about the Analyst software features Access System Documentation The Help guides and tutorials for the instrument and the software are installed automatically Click Start gt All Programs gt AB SCIEX gt Analyst A complete list of the available documentation can be found in the Help e Press F1 to open the Help Contact Technical Support The manufacturer and its representatives maintain a staff of fully trained service and technical specialists located throughout the world They can answer questions about the instrument or any technical issues that may arise For more information visit the Web site at www absciex
38. Tip By using the Define Custom Colors palette users can create customized colors for use in a Contour Plot 1 In the Contour Plot pane click one of the color buttons The Color dialog opens Click a color Click OK The graph changes to reflect the color change Dynamic Background Subtraction Algorithm Dynamic Background Subtraction algorithm improves detection of precursor ions in an IDA Information Dependant Acquisition experiment When the algorithm is activated IDA uses a spectrum that has been background subtracted to select the ion of interest for MS MS analysis as opposed to selecting the precursor from the survey spectrum directly Because this process takes place during LC analysis the algorithm enables detection of species as their signal increases in intensity therefore focusing on detection and analysis of the precursor ions on the rising portion of the LC peak up to the top of the LC peaks maximum intensity Fragment Interpretation The Fragment Interpretation tool helps the user interpret MS MS data Given the chemical structure of a molecule this tool can generate a list of theoretical fragment masses from single Analyst 1 6 1 Software Advanced User Guide 42 of 94 Qualitative Data Analysis non cyclic bond cleavage of that molecular structure The tool can then match the theoretical list with peaks in the current mass spectrum The Fragment Interpretation Tool generates a list of theoretical fragment
39. a file name 4 Click Save Restore Instrument Parameters In the Navigation Bar double click Instrument Optimization Click File gt Restore Instrument Settings Navigate to the instrument settings Click Open SS Ao PU Soe Compound Optimization The Compound Optimization software wizard automatically optimizes an analyte Samples can be introduced using infusion or FIA flow injection analysis The software first checks for the presence of the compounds The voltages of the various ion path parameters are gradually increased or decreased to determine the maximum signal intensity Q1 scan for each ion A text file is generated and then displayed during the optimization process This file records the various experiments performed and the optimal values for each ion optic parameter A file folder containing all the experiments performed is also generated and can be found by opening the data file folder in Explore mode For each experiment performed an acquisition method is also generated and saved in the acquisition method folder Flow Injection Analysis Flow Injection Analysis FIA is the injection of a small quantity of a sample by an autosampler into the LC stream During the FIA optimization process multiple sample injections are performed for various source or compound dependent or both parameter types that are changed between injections FIA optimizes for declustering potential collision energy and collision cell exit po
40. an of zero of the intensity differences The software does not use those pairs of points with an intensity difference larger than 596 of the maximum It sets the noise and area thresholds as follows 3 The noise threshold is equal to the standard deviation calculated in step 4 4 The area threshold is equal to five times the noise threshold Advanced User Guide Analyst 1 6 1 Software 67 of 94 Quantitative Data Analysis calculations produce a value that is lower than this minimum then the software resets Jl Note The minimum value for both the noise and area thresholds is 0 000001 If the the value of that threshold at 0 000001 Recalculate the Noise and Area Threshold If a new background area is defined the software recalculates the noise and area thresholds as follows For each sequential pair of data points the software calculates the standard deviation using a mean of zero of the intensity difference The Analyst software uses all points within the selected range because it is explicitly being told that the selected area is background noise It sets the noise and area thresholds as follows 1 The noise threshold is equal to the standard deviation calculated for the selected range 2 The area threshold is equal to five times the noise threshold Note The minimum value for both the noise and area thresholds is 0 000001 If the calculations produce a value that is lower than this minimum then the software resets
41. as highlights in the fragment list The masses of the two fragments appear on either side of the bond If a spectrum is connected then the Fragment Interpretation tool displays any matching peaks in the graph If a fragment in the list is selected and the fragment is matched to a peak then the Fragment Interpretation window zooms in on that peak View Isotopes The Fragment Interpretation tool can display the theoretical isotopic distribution for a peak matching a fragment in the fragment list 1 oe co N Click Explore gt Show gt Show Fragment Interpretation Tool In the Fragment Interpretation pane click the Options tab Select the Show Isotopes check box Click Apply In the fragment list select a fragment that matches a peak The isotopic distribution for matched peaks is shown in the spectrum Display a Formula Difference in a Spectrum The formula and monoisotopic mass difference between two related hypothetical fragments can be displayed The formula difference is shown when two peaks are selected The formula and monoisotopic mass difference is shown when two fragments are selected or two single non cyclic bonds 1 44 of 94 Click a fragment peak 2 Press the Shift key and then click another fragment peak If the formula difference is equal to a fragment from the fragment list the fragment highlights in the list Otherwise the formula difference between the matching fragments of the peaks is shown in a
42. as this will affect the software functionality BioAnalyst Tutorial Contains a tutorial that guides new users through all the features of the BioAnalyst software A multimedia overview of the BioAnalyst software is also available The folder is available only when the BioAnalyst software is installed Analyst 1 6 1 Software Advanced User Guide 10 of 94 General Information Firmware Contains the instrument system controller software scu21 exe and the instrument firmware files Use these files to download new firmware to the instrument when required For more information refer to the software installation guide included with the software Help Contains the help files guides tutorials release notes and software installation guide Scripts Contains all the scripts that can be installed Research grade scripts are available to extend the functionality of the software Some scripts are installed automatically and some scripts can be installed individually For more information refer to the Scripts User Guide e Simulation Contains the instrument data files required to run the software in simulation mode Projects and Subprojects Decide where to store the files related to the experiment before starting the experiment Use projects and subprojects for each experiment to manage the data better and compare the results Subprojects A subproject contains a subset of the folders in the project All subprojects must conta
43. as well The more constraints that are selected the more precise the list becomes and fewer more relevant matches appear After a set of constraints is defined they will apply to all subsequent searches unless they are edited When a user searches without constraints there is a much larger list of suggested spectra because the library makes fewer specific matches to the spectral data Only peaks above the threshold are used in the search When selecting search constraints The user can also add or subtract peaks from the active spectrum For example if the user thinks a peak is actually a background or noise spike the peak should not be used for the search because it could produce inaccurate results 1 Right click on an active spectrum and then click Search With Constraints The software calculates the centroid of the spectrum automatically Advanced User Guide Analyst 1 6 1 Software 51 of 94 Qualitative Data Analysis Search Constraints Maximum Number of Match 5 Preselect Constraints Preset Tolerance Mass Tolerance af 02 Iba Intensity Factor sp C 1st Precursor m z 7 0 25 C Collision Energy 5 C 2nd Precursor m z 0 25 C Excitation Energy 5 C Retention Time 01 C Record Contains UV Spectrum C Record Contains Molecular Structure Result Sorted by v Comment Contains Keyword Contains Compound Name p Formula Compound Class CAS Number Figure 5 9
44. baseline was found and integrated when the peak was found The types are manual automatic Baseline to Baseline Valley Exponential Skim and Exponential Child IS Signal to Noise The signal to noise ratio of the peak IS Peak Width The ratio of the peak height to its width IS UV Range The UV range of the internal standard IS UV Channel The UV channel of the internal standard IS Peak Width at 50 Percent min A read only column displaying the peak width at 5096 of peak height IS Baseline Slope min A read only column displaying the slope of the baseline IS Peak Asymmetry A read only column displaying the peak asymmetry which is calculated by the following formula Peak End Time Retention Time Retention Time Peak Start Time Values near 1 0 indicate symmetric peaks values greater than 1 0 indicate tailing peaks and values less than 1 0 indicate fronting peaks IS Processing Alg Advanced User Guide A read only column displaying the processing algorithm used Analyst 1 6 1 Software 89 of 94 Quantitative Data Analysis Table 6 12 Internal Standards Column Fields Continued Field Definition IS Integration Quality The Integration Quality Index indicates how well the peak is integrated Values closer to 1 indicate well integrated peaks and values closer to 0 indicate poorly integrated peaks Record Fields The Record fields
45. c layouts and captions that can be opened in Explore mode only These files also contain history information and are similar to data files except that they will contain only the data from the active pane in Explore These files have the pdt extension and are stored in the Data folder in the current project Save a Processed Data File Select the pane Click File Save Processed Data File In the File name field type a name Click Save d e T Open a Processed Data File 1 In Explore mode click File Open Processed Data File 2 Select a file 3 Click Open Qualitative Data The user can view the information contained in a data file in table or graph form Graphical data is shown as a chromatogram or as a spectrum Data in a table is shown as data points The user can perform various sorting operations on the data When the user opens a data file different panes appear depending on the type of experiment performed If the MCA check box is selected in the Tune Method Editor the data file opens with the mass spectrum If the MCA check box is not selected the data file opens the TIC A wiff file can contain data for more than one sample In addition to wiff files the software can open txt files A txt file contains data for only one sample Advanced User Guide Analyst 1 6 1 Software 53 of 94 Qualitative Data Analysis Signal to Noise Ratio The signal to noise ratio is the peak height divided by the noise To
46. com or contact Technical Support using support absciex com Advanced User Guide Analyst 1 6 1 Software Release Date March 2012 7 of 94 AB SCIEX Analyst 1 6 1 Software Advanced User Guide 8 of 94 General Information The software is divided into discrete functional areas called modes Modes allow the user to perform activities related to a main task Modes are accessed through the Navigation bar or the Mode list in the toolbar Users can switch from one mode to another without any loss of work For more information refer to the Analyst software Show Me tutorial CS Analyst E File Edit View Acquire Tools Explore Window Script Help t uy LIP A 5 05605 C acquire Mode HE B Example i kt OR Eau BASS Q i5 D db amp ku M zx Explore Mode GU Configure Quantitate Mode A Security Configuration GH Hardware Configuration X Report Template Editor il Tune and Calibrate AX Compound Optimization AY Instrument Optimization X Manual Tuning fa Acquire SIDA Method Wizard E Build Acquisition Method Build Acquisition Batch ZZ Express View Explore E Open Data File Gg Open Compound Database p Quantitate E Build Quantitation Method N amp Quantitation Wizard Review Results Table L Companion Software Figure 1 1 Analyst Software Window Item Description 1 Mode list 2 Navigation bar Analyst Service The Analyst Service is the communication path between the instrument an
47. d attached devices The Analyst Service is started each time the Analyst software is started In general the Analyst Service starts automatically when the user logs on to Windows If the service is not running when the Analyst software is started then the Analyst Service will start automatically Advanced User Guide Analyst 1 6 1 Software Release Date March 2012 9 of 94 General Information API Instrument Project Folders The API Instrument project folder is important for the instrument to function properly The API Instrument project contains the information required to tune and calibrate the system The API Instrument project folder also contains data files for a manual tune that was performed using the Start button instead of the Acquire button These data files are saved automatically in the API Instrument project folder in the Tuning Cache folder and named with the date and time they were created Empty the Tuning Cache folder regularly The following are some of the folders found in the API Instrument project API BioAnalyst Contains the BTB Data Dictionary csv file This file contains the preset Data Dictionary information required for the BioAnalyst software The appropriate folders appear only when the BioAnalyst software is installed Bundler Contains a program that takes all aspects of a data file wiff file and automatically combines them when the sample is completed e Configuration Contains all the hardware profil
48. d working time on an instrument Create an IDA method with up to two survey scans and eight dependent scans in a single experiment A survey scan is used in IDA to trigger additional experiments The following scan types can be used as a Survey scan Enhanced Multiply Charged EMC Enhanced Product lon EPI second survey scan Enhanced MS EMS e MRM Neutral Loss NL e Precursor lon Prec Q3MS The following scan types can be used as dependent scans EPI e MS MS Analyst 1 6 1 Software Advanced User Guide 28 of 94 Device Methods In an IDA experiment the instrument actions are varied from scan type to scan type based on the data acquired in a previous scan The software analyzes data as it is being acquired and then determines the masses on which to perform dependent scans Uses can set the criteria that will activate an IDA experiment and the method parameters to be used IDA experiments modify experiments and improve results based on the following user defined criteria e lon intensity and charge state e Inclusion and exclusion lists sotope pattern e Dynamic exclusion Rate of change in ion intensity refer to Contour Plots on page 40 Solvent Compressibility Values Table 4 1 Solvent Compressibility Values Solvent Compressibility 10 9 bar Acetone 126 Acetonitrile 115 Benzene 95 Carbon Tetrachloride 110 Chloroform 100 Cyclohexane 118
49. defined views of the Results Table e Full Layout View e Summary Layout View e Analyte Layout View e Analyte Group Layout View Each analyte from a multiple analytes sample can be seen in the Analyte Layout view The preset view is Full layout Full Layout View The preset Full Layout view shows the data for all analytes in the quantitation batch The columns that are displayed depend on the columns selected in the Results Table Columns dialog and the settings selected on the second page of the Quantitation Method Wizard Een Analyte Peak Analyte Peak Analyte Peak Analyte Name Area e Concentration Figure 6 15 Sample Full Layout view Analyst 1 6 1 Software 86 of 94 ia EE serias O blank quus QuanData Wil Peak 2 45e 002 02e 001 0 00 B senes blank Blank QuanData Will Peak k2 1 25e 004 4 53e 003 0 00 3 B seres 0 1 ng ml Standard QuanData Wit Peak 1 7 Ee 002 2 53e4002 0 00 E B series 1ngmL Standard QuanData Witt Peak 2 1 28e4004 4 amp 3e4003 n en B ceras 0 2 npmL Standard QuanData Wit Peak 1 1 55e 203 15 C6e 002 0 00 B senes 0 2 ng mL Standard QuanData Wif Peak 2 1 2Be 10d 4 27 e 003 0 00 B seres 0 5 nc mL Standard QuanData Witt Peak 1 3 Bet 1 Ode 003 0 peu B seres 0 5 nymL Standard QuanDats Wif Peak 2 1 1463004 4 Me 003 ne IB E ceras 1 0 ng mL Standard GuanData wifi Peak 1 7 1260
50. designed to suit different tasks within these modes and then save them for future use When in one of these two modes a particular workspace can be opened without exiting that mode Advanced User Guide Analyst 1 6 1 Software 13 of 94 General Information AB SCIEX Analyst 1 6 1 Software Advanced User Guide 14 of 94 Instrument Tuning and Calibration Tuning the instrument is the process of optimizing the resolution and instrument parameters to attain the best sensitivity and performance of the system Optimizing the resolution means adjusting the peak width and peak shape Users can tune and calibrate the instrument either automatically or manually Automatic tuning The software performs resolution optimization and mass calibration using the Instrument Optimization wizard For linear ion trap LIT instruments MS3 optimizations are also performed Manual tuning Users can perform many of the instrument resolution optimizations and calibrations manually Automatic Tuning and Calibration Instrument Optimization is automatic instrument tuning software that tunes both quadrupole and LIT modes and performs mass calibration For quadrupole mode it adjusts the resolution offsets For LIT mode it optimizes AF3 and EXB For MS3 it adjusts the excitation and isolation coefficients Select one of the instrument performance options e Verify instrument performance Tests the instrument performance but leaves the instrument settin
51. display additional information about each sample record information that is applicable only to the analyte not the internal standard Table 6 13 shows the available fields Table 6 13 Record Fields Field Definition Use Record Indicates whether this record should be included for calibration Applies to standards and QCs If the check box is cleared then the unused standards and QCs are struck out in the Statistics Table Record Modified Indicates whether the quantitation method used for the record was modified in any way from the original Calculated Concentration The calculated concentration of the analyte as calculated using the calibration curve Relative Retention Time The ratio of the retention times of the internal standard and the analyte Accuracy The calculated concentration divided by the known concentration as a percentage Response Factor The peak area or height depending on the regression option divided by the analyte concentration Sample Column Fields The Analyte Results fields display information about each analyte and internal standard if one was used after analysis Table 6 14 shows the available fields Table 6 14 Results Tables Sample Column Fields Field Definition Analyte Peak Name The name of the analyte Analyte Units The units in which the analyte concentrations are given Analyte Peak Area The area of the analyte Analyte Peak Height The height
52. djustment Analyst 1 6 1 Software Advanced User Guide 60 of 94 Quantitative Data Analysis Analyst Classic and IntelliQuan Integration Algorithms The IntelliQuan algorithm uses one of two peak finding parameters Automatic IQA II which is a parameterless setting or Specify Parameters MQ III After integrating peaks using the IntelliQuan algorithm choose which peak finding parameter best fits the data set This is done in the displayed peak integration parameters in the Peak Review pane or window Table 6 3 shows the parameters available with the Analyst Classic algorithm Table 6 3 Analyst Classic Algorithm Parameter Default Bunching Factor Definition The number of points to be averaged together and considered as a single point for the purpose of peak finding Default Number of Smooths The number of times to smooth the chromatogram Default Void Volume Retention Time Any peaks that appear before this time are ignored Default Concentration Units The concentration units used to describe the sample concentration for example pg uL Default Calculated Concentration Units The concentration units used to describe the calculated sample concentration for example pg uL Default RT Window The time window centered at the expected retention time for peak finding For example a 30 second retention time window gives an additional 15 seconds before and after the expected retention time
53. e The centroid algorithm converts peaks to single values by using an intensity weighted average to calculate the center of gravity of the peak The output of the algorithm is a list of peaks with parameters as shown in Table 5 1 Table 5 1 Peak Parameters Parameter Definition Centroid Value The value of the centroided data in units of mass or time Intensity The intensity of each peak in cps Width The width of the centroided peak in amu Data is automatically calculated as a centroid when added to a library or when a search is conducted Calculate the Centroid of a Peak 1 Select a pane containing a spectrum Calculating the centroid of the peak changes the appearance of the existing graph To compare the result with the original data make a copy of the graph before calculating the centroid 2 Click Explore Centroid Analyst 1 6 1 Software Advanced User Guide 36 of 94 Qualitative Data Analysis Centroid Location Time Figure 5 2 Analyte Centroid Location Data Analysis Users can open files containing existing data or data that is currently being acquired All experiment related data can also be viewed in tabular form The table pane consists of two tabs the Data List tab and the Peak List tab The Data List tab contains experiment related information such as acquisition time and scan intensity The Peak List tab displays peak related information such as peak height peak area and bas
54. e Versus Flow Rate 0 ccc ellen 29 Advanced User Guide Analyst 1 6 1 Software Release Date March 2012 3 of 94 Chapter 5 Qualitative Data Analysis leeren 31 Chromatograms s scs deteqec Soo e Ede Rome a Cu d aes Reda ura 31 Spectra saner e o WR C WA EE WI a odes Data Cea od e C RECS OR Ae RO 32 Background Subtraction eed RR E Oh Ree need Ew ene BERR EG OS 32 Perform a Background Subtraction from a Spectrum 32 Unlock Ine Ranges vd ese x es dn ette p eq el x Ea 33 Background Subtract to File 4 uda des ru d thedore Mae TES EA TE 33 Perform a Background Subtraction to File 2 22005 33 Baseline Subtract uu cot mco oco ox eR babe teda boe E Rt ets 34 Calculator S ot sies deed EE ttes Soie ode E A a epee 35 Elemental Composition Calculator 0 0 ccc cen 35 Hypermass Calculator 52534 M med wu E EE ork nants Bleek al ai 35 Elemental Targeting Calculator l l 35 Mass Property Calculator da 4 3 ev VERE ath eas Bae beet e ERE E ele 36 Isotopic Distribution Calculator 0 0000 eee 36 Centroided Peaks ora dis eo ted ERE ened Kier e t Wwe Ah rs 36 Calculate the Centroid of a Peak 0002 eee 36 Data Analysis urn c giiatom eels ee USUS Boedo CR Baud 802 Av Ooo abun Shue 37 Total lon Chromatogram ses ks Rer e Shee died ae ER shares 37 Extracted lon Chromatogram 0 ce eee eee 37 Base Peak Chromatogram 00002 cece
55. e mass intensity time and collision energy of ions on which product ion scans were performed in either a list view or a tree view In list view the list can be sorted by double clicking on any column header Use the Appearance Options dialog to customize the columns in the list view On the right side the viewer is split into four panes The top left pane displays the survey TIC data The bottom left pane shows the XIC of the mass The top and bottom right panes show the survey and product scans respectively The IDA viewer lists all the masses on which Enhanced Product lon scans or Enhanced Resolution scan types were performed In the IDA viewer users can do the following e Click a mass in the list or tree view to display plots relevant to that mass View the survey spectrum from which the mass was identified and the product spectrum of that mass Display the TIC of the survey scan and the XIC for each mass contiguous across a number of cycles When a merged mass is displayed it indicates 7 Note Brackets around a mass indicate that the mass is merged A merged mass is an averaged spectrum containing the average of all contiguous spectra Advanced User Guide Analyst 1 6 1 Software 45 of 94 Qualitative Data Analysis Mass Tolerance 0 100 Da lu L Rd aul OF Mass Window Da 5 000 m Mass List List View TIC of EMS Exp x WB ER Exp 2 0 013 m Max 0 0 c Centoidm Timefmin Scan ct z E o7 ER
56. e peak users may not agree with the start or end points selected Peak Review Tips Table 6 6 Peak Review Tips To do this Peak Integration To review peaks do this To review all peaks make sure that all samples are listed in the Results Table The Peak Review window contains the peaks listed in the Results table If some samples are hidden in the table for example if a query is applied then they are also hidden in peak review Peak Integration To move to the first peak in the batch Detect Peaks Right click anywhere in the Peak Review pane and then click Show First Page To move to the last peak in the batch right click anywhere in the Peak Review pane and then click Show Last Page The software detects peaks in four stages 1 First it finds the potential peak start by examining the distance between each bunched point and the preceding one When the distance exceeds the current noise threshold a potential peak start has been found 2 Thenit confirms the peak start by making sure that enough points exist in a row to exceed the area threshold 3 Next it finds the peak top by searching for a point that is lower than the previous point 4 Finally it finds the end of the peak by identifying the place where the distance between one bunched point and the next falls below the noise threshold If necessary it then separates peaks Advanced User Guide Analyst 1 6 1 Software 69
57. e rate Al is changed the software automatically calculates the interval again 3 Dothe following to set the channel details Advanced User Guide Analyst 1 6 1 Software 25 of 94 Device Methods e Inthe Channels field click the channel name and then select the check box beside the name to include it in the method Inthe Interpreted Value Full Scale field type the appropriate value Inthe Interpreted Unit field type the appropriate unit The number of available channels is specified when setting up the ADC in the hardware profile 4 Savethe file Analyst 1 6 1 Software Advanced User Guide 26 of 94 Device Methods Dynamic Fill Time Dynamic Fill Time DFT is a feature specifically designed to optimize the data obtained in every spectrum for the linear ion trap scan functions DFT will automatically adjust the fill time used to fill the ion trap based on the ion flux coming from the source For more intense ions the fill time will be automatically reduced to ensure the trap is not overfilled with ions For less intense ions the fill time will be automatically increased ensuring that good ion statistics are obtained in the spectrum DFT is applicable for the following scan types e Enhanced MS EMS Enhanced Resolution ER Enhanced Product lon Scan EPI e MS MS MS MS3 Adjust the DFT settings by selecting Tools Settings Method Options in the software Experiments and Periods The mass spectro
58. e setting options procedures can be table specific or global Table specific settings When table settings are modified on a table itself the changes to the settings are available only to that table however they can be exported as global settings Global settings Modifying global settings involves making changes to a group of settings that can be applied to future Results Tables To customize a Results Table that is being created choose a group of settings on the Create Quantitation Set Select Settings amp Query page If a group of settings is not selected the software automatically uses the preset settings How Accuracy Variations Affect the Results For preset queries accuracy is expressed as a percent and implemented as plus or minus that number For example if 10 is typed for Maximum Variation for standards in the Create Default Query dialog then all records containing standards whose accuracy falls outside 90 and 110 are displayed in the Results Table If 5 is typed only standards whose accuracy is less than 9596 and greater than 10596 would appear in the Results Table For more information refer to Results Tables on page 82 Regression This section gives the equations for each of the regression types In the following equations x y and w are as described in Weighting Factors on page 94 In the following equations x represents the analyte concentration for standard sample types and y represents the corresponding
59. e the acquisition method by adding or removing device methods If the required device icon is not in the Acquisition Method Browser pane then users can add the device only if it is included in the active hardware profile Devices in Acquisition Methods Create an acquisition method for a device by selecting the operating parameters for that device Acquisition methods can be created for any of the following devices if they are configured in the active hardware profile Pumps Autosamplers e Syringe pumps Column ovens e Switching valves Diode array detector Analog to digital converters Integrated systems For information about setting properties for devices refer to the Peripheral Devices Setup Guide manufacturer al Note The available parameters for the LC devices vary depending on the Add or Remove an LC Device 1 With a method file open in the Acquisition Method Editor in the Acquisition method pane right click Acquisition Method and then click Add Remove Device Method Advanced User Guide Analyst 1 6 1 Software Release Date March 2012 23 of 94 Device Methods Add Remove device methods v OTRAP 4500 v Shimadzu LC Controller Method Agilent 1290 G4212A DAD Method Figure 4 1 Add Remove Device Method dialog 2 Select or clear the check boxes beside the device method to add or remove the device method 3 Click OK Set the LC Pump Properties 1 With an acquisition method file
60. ee eee eee 37 Extracted Wavelength Chromatogram eee ee eee 38 Diode Array Detector 0 2 2 00 e eee 38 Total Wavelength Chromatogram 0 0 0 eee ee 38 Ovellay Graphis i te tO ek anc ne iO Sg EN IAE p prt 38 Cycle Between Overlaid Graphs 000 2c eee ees 39 Um OVer d S chs atte buste Em b darte xupc iUm eder a Hes bas de rich tees 39 Graphs Labels xis o erc ee RR o e ORO aie DR ar RE Re up ras 39 Add Captions to a Graph s ieoten a E E A EE lees 39 Add Text to a Graph 2000s 40 Compound Database 0 00000 eee 40 Contour PIOUS 345 25 Seance Tecate ee Seales woo ms Gea RH eerie dee Wd det ede 40 View a Contour Plot duerme bh ETE hatte wield rere AS 41 Select an Area in a Contour Plot lllillleleeiieleeeees 41 Set the Intensity and Absorbance in a Contour Plot 42 Change Colors in a Contour Plot 0 0 0 0 0 eee 42 Dynamic Background Subtraction Algorithm llli 42 Fragment Interpretation ex dees teeows pibe E ERE RE Bets EE d rA NE 42 Connect the Fragment Interpretation Tool to a Spectrum 43 Match Fragments with Peaks 45550 e ee eee eee eie 44 Select a Bond in a Molecular Structure 0 00 cee eee ee 44 View Isotopes ovx Ves ed ee Parke A ERA a 44 Display a Formula Difference in a Spectrum 2 2000055 44 Display a Formula Difference in the Fragment List
61. eline type Total lon Chromatogram A Total lon Chromatogram TIC is created by summing the intensity contributions of all ions from a series of mass scans Users can use the TIC to view an entire data set in a single pane It consists of the summed intensities of all ions in a scan plotted against time in a chromatographic pane If the data contains results from multiple experiments individual TICs Total lon Chromatograms for each experiment and another TIC that represents the sum of all experiments can be created The preset TIC that represents the sum of all of the experiments is shown with a splitter tool below the center of the x axis Extracted lon Chromatogram An Extracted lon Chromatogram XIC is an extracted ion chromatogram created by taking intensity values at a single discrete mass value or a mass range from a series of mass spectral scans It shows the behavior of a given mass or mass range as a function of time The intensity of the ion or the summed intensities of all ions in a given range is plotted in a chromatographic pane Base Peak Chromatogram A Base Peak Chromatogram BPC displays the intensity of the most intense ion in every scan as a function of scan number or retention time It is useful in instances where the TIC is so dominated by noise that there is a large offset and chromatographic peaks are hard to distinguish It is also helps to distinguish between co eluting components BPCs only be generated from s
62. er tab 3 In the Available Libraries section click New Analyst 1 6 1 Software Advanced User Guide 48 of 94 Qualitative Data Analysis 4 Type a name for the library 5 In the Database Information section select MS SQL Server server Add Library Library Information Enter a Name for the Library Database Information Database type 7 MS Access local MS SOL Server server Enter the name of the database server Te Enter the name of the database on the server Security Information Use Windows integrated security Use a specific user name and password User Name Password Save Cancel Figure 5 7 Add Library dialog 6 Type the name of the database server 7 Type the name of the database 8 Doone of the following Ifa specific user name and password are required to access this database then type the user name and password f Windows security is used then in the Security Information section select the Use Windows integrated security option 9 Click Save View All Library Records e Click Explore gt Library Search gt List The Librarian dialog opens with all records in the database Add a Record to the Library 1 Right click an active spectrum and then click Add a Record Advanced User Guide Analyst 1 6 1 Software 49 of 94 Qualitative Data Analysis The spectrum is automatically calculated as a centroid The Add a Record dialog opens with data
63. ersus its area or height or ratios if an internal standard is used The area or height of a sample is then applied to this curve to determine the sample concentration as displayed in the Results Table The regression equation generated by this calibration curve is used to calculate the concentration of the unknown samples The software places the known concentrations or ratios on the x axis and the calculated area or height or ratios on the y axis It then plots the points for all the standards in the batch The system produces a best fit curve to those points through regression and weighting type that is selected This curve is used along with the area or height for the unknowns to interpolate the concentration Select the Best Regression Type After selecting a regression type fit the user cannot see the calibration curve from the wizard Instead use the preset values experience or corporate policy to choose a regression type After changing the fit check the Accuracy column in the Results Table for changes The better the fit is the better the accuracy of the quantitative analysis will be The calibration curve plots the standards concentration against its peak area or height or ratios if an internal standard is used When the points for the standards are plotted determine the best fit for the curve to these points and indicate the choice in the Specify Calibration dialog of the wizard The preset fit is linear which assum
64. es The minimum values on either side of the current data point minima within the window are located A straight line is fitted between the two minima and the height intensity of the current data point above the line is calculated The end points of the data are regarded as minima e The data point is replaced with the new calculated value Analyst 1 6 1 Software Advanced User Guide 34 of 94 Qualitative Data Analysis Calculators Users can perform calculations on the basis of collected data Although the calculator is a separate window it is connected to the active graph within the software The following calculators are available Elemental Composition Calculator e Hypermass Calculator Elemental Targeting Calculator e Mass Property Calculator e Isotopic Distribution Calculator Users can cut and paste from one text box to another between the different windows in the calculators Data from any of the calculators can be printed by clicking the Print icon in the top left corner of the window For more information about using calculators refer to the Help Data from the Elemental Composition Mass Property and Isotopic Distribution calculators can be exported to a separate file Use the Elemental Targeting calculator to modify the data within the active graph Data from the HyperMass and Isotopic Distribution calculators can be overlaid on the active spectrum Tip Setthe precision of calculator data in the Calc
65. es hwpf files Example Scripts Contains some of the scripts that are used with the software Instrument Data Contains a file called InstrumentData ins The file stores all the critical calibration information and more Method Tables Contains all instrument parameters that define the enhanced scan functions Do not change the files in this folder Changing the contents of this folder will affect the performance of the enhanced scan modes Parameter Settings Contains all the instrument parameters and linkages Instrument parameters are saved as ParamSettingsdef psf files Preferences Contains the Tunedata tun file All settings parameter tuning instrument processing appearances and queue are saved as Tunedata tun in this folder e Processing Scripts Contains the scripts for data processing in Explore mode Scripts are found in the Script menu Queue Data Contains information from the queue e Tuning Cache Contains all the data created in Manual Tuning by clicking Start instead of Acquire Files are saved with a generic time and date stamp for their names The Tuning Cache folder holds a limited number of files and will overwrite files as needed Save the files with a new name and move the files immediately if they need to be saved Program Files The following folders are found within the Program Files Analyst folder bin Contains the software program files Contents of this folder should not be changed
66. es All of the sets in a batch use the same hardware profile however samples in a set can have different acquisition methods A batch can be submitted only from an acquisition station Batches link together e Sample information such as name ID and comment e Autosampler location rack information Acquisition methods Processing method or script optional e Quantitation information optional Custom sample data optional e Set information Batch Editor Use the Batch Editor to create or modify batches and to create batch templates To run samples each using different acquisition methods select multiple acquisition methods in the same set An acquisition method can also be used as a template In this case the same method is used for each sample but the user can select different masses or mass ranges for each sample The Batch Editor can also be used to import sample lists created in external programs such as Microsoft Excel The user can modify every detail of the batch before submitting it for processing When a batch is submitted for analysis the user can submit the entire batch specific sets within the batch or specific samples within a set For example to analyze ten samples five using one acquisition method and five using a different acquisition method create a batch of two sets one for each method used Table 3 1 Batch Editor Tabs Tab Description Sample Used to create the sample list and to select
67. es that all the standards will fall on a straight line Select from the types of fit in Table 6 1 Table 6 1 Types of Fit Fit Description Linear regression assumes that the standard points fall on a straight line Advanced User Guide Analyst 1 6 1 Software 20 59 of 94 Quantitative Data Analysis Table 6 1 Types of Fit Continued Fit Description Linear Through Zero Linear Through Zero regression assumes that the standard points fall on a straight line and that the points line up with the zero point on the x and y axes Use this setting to force the line to go through the zero point Quadratic If the standard points do not fall on a straight line use quadratic regression to produce a quadratic fit to the data points Mean Response Factor If the standard points fall on a straight line then to average the points use mean response factor regression to produce an average of the slope for every point on the curve Power If there is some linear and some curvature in the line of points use power regression instead of linear or quadratic regression to produce a line somewhere between these fits Choosing the Best Weighting Factor The calibration curve plots the standards concentration against its peak area or height When the points for the standards are plotted determine the best weighting factor for these points and indicate it in the Specify Calibration dialog The preset fit is None which ass
68. ft key and then select another background range To set the subtract range click Explore gt Background Subtract gt Set Subtract Range 5 Select the peak of interest Analyst 1 6 1 Software Advanced User Guide 32 of 94 Qualitative Data Analysis 6 Click Explore Background Subtract Perform Background Subtract The background is subtracted from the peak and a new spectrum is generated 7 Toisolate another peak drag the locked ranges in the chromatogram and repeat the background subtract Tip To clear the background subtract region click Explore gt Background Subtract gt Clear Subtract Range Ed 8 To save the background subtracted spectrum as a processed data file click File gt Save Unlock the Ranges The selected subtraction range is set to locked Click Explore Background Subtract Subtract Range Locked The ranges are unlocked and each one can be moved independently Background Subtract to File Use Background Subtract to File to save a new file that has a defined noise region subtracted from each scan When a range is selected in the TIC all the scans from that selection are internally averaged and the resulting spectrum is subtracted from all the scans Use the Background Subtract to File dialog to select where the file is to be saved In some cases the resulting file might look close to the original scan Perform a Background Subtraction to File 1 Open a data file 2 Selec
69. g 4 Doone of the following To perform the sort save the sort criteria and close the Sort dialog click Save Execute To perform the sort and close the Sort dialog without saving the sort criteria click Execute Save Default Sort Criteria for Future Results Tables 1 Click Tools Settings New Quantitation Results Table Settings Table Settings New Table Settings for Proj Default PK Data Settings Figure 6 13 Table Settings dialog 2 Expand the Table Settings folder and then double click the Default folder 3 From the expanded Default folder select the Sorts folder 4 Click New Analyst 1 6 1 Software Advanced User Guide 84 of 94 Quantitative Data Analysis Name Sort By Group v Ascending Column v Descending Then By Group v Ascending Column w O Descending Then By Group v Ascending Column v O Descending Figure 6 14 Sort dialog In the Name field type a name For each sorting rule to be set in the Sort By section do the following i Inthe Group list select the type of column ii In the Column list select the column iii Select the direction of the sort Ascending or Descending To save the criteria and close the Sort dialog click OK Click Done Sort a Results Table using Preset Sort Criteria Right click in the Results Table click Sort and then select the name of the sort About using Quer
70. g criteria can also be edited To view the problem sample try plotting the concentration of the unknown against time or plotting the area of the internal standard against the index Analyst 1 6 1 Software Advanced User Guide 66 of 94 Quantitative Data Analysis Save Default Plot Criteria for Future Results Tables 1 Right click in the Results Table and then click Table Settings gt Export To New Table Settings This will export the table settings from the rdb so that it can be reused in other quantitation runs within the project 2 To export table settings to another project click Tools Project Copy Data Project Source Directory C Analyst Data Projects Source Project Name Example Target Project Name Tutorial2011 06 26 Directories Acquisition Methods C Quantitation Methods C Report Templates Table Settings Noise and Area Threshold Parameters To identify peaks the software requires a set of noise and area threshold parameters The software sets these parameters initially but they can be changed later It sets the parameters as follows 1 The software calculates the largest intensity difference between any two sequential data points This number represents the difference between two intensities not the actual intensity itself 2 For each sequential pair with an intensity difference of less than 5 of the value calculated in Step 1 it calculates the standard deviation using a me
71. gs unchanged A report is generated at the end of the test Use this option weekly to check how well the instrument is performing Adjust mass calibration only Automatically checks and adjusts the mass calibration If the mass calibration has changed then the software corrects it Use this option weekly for LIT instruments or monthly to check and adjust the mass calibration if required Adjust instrument settings Checks and adjusts the instrument settings and mass calibration The instrument settings are updated from the current settings to optimal settings Use this option if instrument performance is poor or if the peak shape is bad Only experienced users should adjust the instrument settings e Reset selected scan modes to default values and adjust instrument settings Resets the instrument values to the factory preset values Select this option if a major component of the instrument is replaced or after the first installation Only FSEs should use this feature Advanced User Guide Analyst 1 6 1 Software Instrument Tuning and Calibration Back up Instrument Parameters Back up the current instrument parameters in case they must be restored later The preset location for the instrument parameters is C Analyst Data Projects VAPI Instrument Instrument Optimization Instrument Settings Backups User Created Backups 1 In the Navigation Bar double click Instrument Optimization 2 Click File Backup Instrument Settings 3 Type
72. hich the purchaser may put the equipment described herein or for any adverse circumstances arising therefrom For research use only Not for use in diagnostic procedures The trademarks mentioned herein are the property of AB Sciex Pte Ltd or their respective owners AB SCIEX is being used under license AB SCIEX 71 Four Valley Dr Concord Ontario Canada L4K 4V8 AB Sciex LP is ISO 9001 registered 2012 AB SCIEX Printed in Canada REGISTERED COMPANY Contents Ibo ic cies cee eae eee dees Gila a ee wees Dae ee He Wed alee Wes 7 Access System Documentation 0 0 0 cece eee 7 Contact Technical Support 5 oca e ka Ro Wea e e dc i ec ot ok e n a 7 Chapter 1 General Information 0 00 cece eee ee 9 Analyst Service METER TERT 9 API Instrument Project Folders 0 0 00 c ee eee eee 10 chm m P x 10 Projects and Subprojects ua saubidees da Seba e abo de score Do dead o RR a 11 zuo APP 11 Project Organization au ck 3C ed RC AORGCACRCR ACA RARER RED Es ER A 12 Software Security 2 evade ee ONG de Rok ACRI E Ra ROCA CER ORC E NR aon og Rd 13 Workspaces icxesaaw e e t RO ACC e ROR Re ea 13 Chapter 2 Instrument Tuning and Calibration 200 eee e eee 15 Automatic Tuning and Calibration 0 0 0 ee 15 Back up Instrument Parameters 0 00 ee nes 16 Restore Instrument Parameters 0 00
73. ibing the sample Set Number A number that identifies a subset of an entire batch Acquisition Method The name of the method used to acquire the samples Acquisition Date The date and time when the acquisition was run Rack Type Analyst 1 6 1 Software 92 of 94 An identifier associated with the kind of autosampler rack used if any Advanced User Guide Quantitative Data Analysis Table 6 15 Sample Column Fields Continued Field Rack Number Definition The rack position in which the sample was placed when it was acquired In single rack autosamplers this is always 1 Vial Position The position in the autosampler plate where the vial was located Plate Type An identifier for the kind of plate used multiplate racks only Plate Number The plate position on the rack multiplate racks only File Name The name of the raw data file This name is not unique because the data from many samples may be contained in a single data file Dilution Factor The amount by which the sample was diluted used to determine the calculated concentration Sample Annotation Additional comments describing the sample Weight to Volume Ratio Table 6 16 DAD Fields Gives the weight to volume ratio for the sample Field Analyte Peak Area for DAD Definition The area of the analyte peak mAU min Analyte Peak Height for DAD The height of the analy
74. ies with Results Tables A query is a request for records in a Results Table that the certain conditions set using textual or mathematical selection criteria Apply a query either during the process of generating a Results Table or after one has been generated These two types of queries are called default and table specific queries For more information refer to Default Queries and Table Specific Queries on page 75 Compare Results Between Batches When more than one Results Table is displayed obtain statistical information about the standards and QCs for additional batches in the Statistics window Normally results are Advanced User Guide Analyst 1 6 1 Software 85 of 94 Quantitative Data Analysis compared between batches to look for trends in the standards or QCs or to verify that the method is valid For two or more open Results Tables compare results in the Statistics window Both sets of statistics appear in the Statistics window How Concentration Levels Affect Results Note The number of analytes and the number of analyte names must be the same for the data to be combined in the Statistics window The concentration is defined for all QCs and standards If there is a change in the accuracy of the concentration level by more than the amount defined in the Max Variation field in the Create Default Query dialog then this information is shown in the Results Table Results Table Layouts The software has four pre
75. in the same folders Subprojects are useful for organizing data For example if samples of various compounds from different laboratories are run using the same acquisition method then create subprojects to store the results for each laboratory but leave the acquisition method folder in the project The acquisition method is then available for use in the subproject or laboratory Alternatively if samples are being analyzed over a period of several weeks then the results from each day can be stored in a separate subproject Refer to Figure 1 2 Example of a project and subproject folder structure Advanced User Guide Analyst 1 6 1 Software 11 of 94 General Information Cid Analyst Data ED Projects C API Instrument 4 9 Default 9 Example B C My Experiment Ca Acquisition Methods E Acquisition Scripts Processing Methods E Processing Scripts C Project Information C Quantitation Methods GD Sample A H Batch C Data CJ Log E Results Sample B H Batch CJ Data C3 Log E Results C Sample C H Batch Ld Data 9 Log E Results E Templates H Workspaces F Figure 1 2 Example of a project and subproject folder structure Project Organization A project is a folder structure for organizing and storing sample information data quantitation information and so forth Within each project there are folders that can contain different types of files for example the Data folder contains acquisition data files Table 1
76. ingle period single experiment data Advanced User Guide Analyst 1 6 1 Software 37 of 94 Qualitative Data Analysis The graph uses two colors alternating each time the mass of the base peak changes The color changes are maintained when the data is manipulated by scrolling or zooming For information about selecting the colors used in the graph refer to the Help Extracted Wavelength Chromatogram An Extracted Wavelength Chromatogram XWC is a wavelength chromatogram created by taking intensity values at a single wavelength or by the sum of the absorbance for a range of several wavelengths Diode Array Detector Users can view the Diode Array Detector DAD spectrum for a single point in time or for a range of time as a Total Wavelength Chromatogram Total Wavelength Chromatogram A Total Wavelength Chromatogram TWC is a less commonly used chromatogram It displays the total absorbance mAU as a function of time The TWC provides a way of viewing an entire data set in a single pane It consists of the summed absorbances of all ions in a scan plotted against time in a chromatographic pane If the data contains results from multiple experiments individual TWCs for each experiment and another TWC that represents the sum of all experiments can be created Overlay Graphs Two or more sets of data can be visually compared by overlaying graphs created by similar methods Each individual spectrum is distinguished by the color of its t
77. is presented either as a chromatogram or as a spectrum Data from either of these displays can be viewed as a table of data points and various sorting operations can be performed on the data The software stores data in files with a wiff extension Wiff files can contain data for more than one sample In addition to wiff files the software can open txt files txt files contain data for only one sample When a data file is opened in the software different panes appear depending on the type of experiment that was performed If the MCA check box is selected in the Tune Method Editor the data file will open to the MS mass spectrum If the MCA check box is not selected then the data file opens with the Total lon Chromatogram TIC Users can select a range and then double click in the TIC pane at a particular time to show the MS for this range Chromatograms A chromatogram displays the variation of some quantity with respect to time in a repetitive experiment for example when the instrument is programmed to repeat a given set of mass spectral scans several times Chromatographic data is contiguous even if the intensity of the data is zero Chromatograms are not generated directly by the instrument but are generated from mass spectra In the chromatogram display the intensity in counts per second cps is shown on the y axis versus time on the x axis Peaks are automatically labeled In the case of LC MS the chromatogram is often dis
78. is saved as a new data file For more information refer to the Help Subtraction Use the subtraction option to subtract a control or background from a sample Addition Use the addition option to add separate chromatograms together to create a single more complete profile for a compound For example create several chromatograms using different collision energies and then add the chromatograms together to create a single profile Average Use the average option to statistically improve the profile for a compound and to generate more reliable spectra Toolbar Icons Table 5 4 Toolbar Icons Icon Name Function Perform Background Performs a background subtract after the Subtract background ranges have been selected Subtract Range Locked Locks the selected background ranges If the background ranges are unlocked then users can move each range independently Background Subtract to File Saves a new file that has a defined noise region subtracted from each scan Be E e Spectral Arithmetic Wizard Uses the Spectral Arithmetic Wizard to perform arithmetic operations using two complete chromatograms to create a new chromatogram which users can save as a new data file 56 of 94 EE Centroid Calculates the centroid of the data iL a Home Graph Returns the graph to the original scale Analyst 1 6 1 Software Advanced User Guide Qualitative Data Analysis Table 5 4 Toolbar Icons Continued
79. k in the Results Table and then click Metric Plot New Advanced User Guide Analyst 1 6 1 Software 65 of 94 Quantitative Data Analysis 10 Metric Plot Save Execute Cancel Name A Axis Group Index Execute Column Help Y Axis Group Index Column Show Regression None v Weighting Percent Multiplier Figure 6 2 Metric Plot dialog In the Name field type the name for the new plot criteria In the X Axis section in the Group list to plot a field in the y axis using the x axis as an index select Index and leave the Column list blank If required to plot two columns against each other in the Y axis section in the Group list select Internal Standard and then in the Column list select IS Peak Area If required in the Regression list select the type of regression to be used and then select the appropriate regression settings To generate the plot and save the plot criteria click Save Execute The metric plot opens For more information refer to Figure 6 1 Example of a metric plot on page 65 Right click in the plot pane and then click Data Legend to view an explanation of the colors used by the plot Right click in the plot pane and then click Point Legend to view an explanation of the symbols used by the plot This set of criteria is now available for future plots of this Results Table Right click in the Results Table to access the criteria The plottin
80. k the relationship and then click And or Or 9 To exclude compounds containing a certain number of atoms of specific elements select or type the elements in the Elements Included table and then type a minimum and maximum number of atoms of the element Note Element symbols are case sensitive For example Hydrogen is H Al not h and Sodium is Na not NA or na p P 10 To exclude compounds containing certain elements select or type the elements in the Excluded table 11 To search for compounds fitting the criteria click List Records that match all the constraints appear in the Records table Listing constraints are saved Library Search Tips Table 5 3 Library Search Tips To do this do this Group conditions Select the conditions to group and then click Group This function behaves like parentheses in formulas Search without using Right click an active spectrum and then click Search Library constraints The Search Results dialog opens Search for a Similar Spectrum The user can search the library for a spectrum and its related compound information that matches or is similar to an active spectrum Searches can be performed with or without constraints When the user searches with constraints only those records that match all the criteria appear The results appear in a ranked list the first item on the list is the best fit to the active spectrum Entries lower in the list do not match
81. ld a Batch as a Text File Users can export a batch only if it contains at least one set with at least one sample If the text file is saved it can be used again later as a template 1 Make sure that the active hardware profile includes all the devices needed to acquire the samples In the Navigation Bar under Acquire double click Build Acquisition Batch Create a single set single sample batch Click File Export Name the file Click Save Open the text file in a spreadsheet program oN Oa oO DN Type or copy and paste the details for the samples one sample per row with the details under the appropriate headings Note Do not delete any of the columns The columns in the spreadsheet An must match the columns in the Batch Editor 9 Save the modified text file as a txt or csv file 10 Close the spreadsheet program The text file can now be imported into the Batch Editor Import a Batch as a Text File Before batch information is imported from a text file make sure the data in the file is organized and formatted correctly In particular the column headings in the spreadsheet must match the Batch Editor column headings 1 In the Navigation Bar under Acquire double click Build Acquisition Batch Analyst 1 6 1 Software Advanced User Guide 20 of 94 Batches In the Sample tab right click and then click Import From gt File Click the text file containing the batch information Note If
82. lope is calculated as m Ewxy Xwx2 The correlation co efficient is calculated as r Ewxy J Zwx2Xwy2 If the standard points fall on a straight line then to average the points use mean response factor regression to produce an average of the slope for every point on the curve Mean Response Factor The mean response factor calibration is calculated as y mx Advanced User Guide Analyst 1 6 1 Software 77 of 94 Quantitative Data Analysis This is the same equation as for the linear though zero case However the slope is calculated differently as m Xw y x XZw The standard deviation of the response factor as c J nD n 1 Ew Where D EXw Xwy2 x Ewy x b Note Points whose x value is zero are excluded from the sums If there is some linear and some curvature in the line of points then use power regression instead of linear or quadratic regression to produce a line somewhere between these fits Power The power function calibration equation is calculated as follows The equations for the linear calibration are used as described previously to calculate the slope m and intercept b except that x in those equations is replaced by In x and y replaced by In y When this is done a p and the correlation co efficient r are calculated as a exp b p m r r If the points of the standard do not fall on a straight line use quadratic regression to produce a quadratic fit to the data points This setti
83. low the user to select the batch or batches to be quantified create or select a quantitation method and then integrate the sample data The difference between the two is the type of method created The standard wizard creates a standard method while the automatic wizard creates an automatic method Peaks are not verified as part of the method creation the peaks can still be reviewed after integration has taken place There is only one common occasion for which peaks do not need to be verified when quantitation is done simply to integrate and not to find concentrations This may need to be done for example for a batch that contains different compounds in every sample or when the mass is not the same from sample to sample If this is the case use the automatic wizard Otherwise to perform quantitation use the standard wizard Use the Standard Quantitation wizard after acquiring the sample to do the following Choose arepresentative sample Analyst 1 6 1 Software Advanced User Guide 62 of 94 Quantitative Data Analysis e Select analyte and internal standard peaks e Adjust peak finding and integration parameters e Review peaks during method creation e Select calibration Use the Automatic Quantitation wizard to select a batch create a method without peak confirmation and then integrate the sample data This wizard is quicker than the Standard wizard and does not require that the masses scanned be the same for all sample
84. m of area slices greater than threshold ennt o No Figure 6 5 Confirming the actual peak start Item Description 1 Look through data points in this region 2 Minimum data point 3 Potential peak start Find the Peak Top To find the peak top the software first looks for a point that is lower than the preceding point Then to confirm that it has found the top correctly it sums the intensity differences between the potential top and subsequent bunched points until it reaches the end of the peak If the total distance between points exceeds two thirds of the area threshold then the peak top is confirmed That is the software makes sure it has a peak first and then works backward to find the top of it Advanced User Guide Analyst 1 6 1 Software 71 of 94 Quantitative Data Analysis If however the software finds a higher bunched point before the area test has been passed then it identifies a new top and restarts the area test described previously Instead it is determined from a quadratic fit based on the three Note The actual retention time for a peak is not simply the point identified as highest data points a Figure 6 6 Finding the peak top Item Description 1 Sum of area slices is greater than two thirds of the area threshold DN Figure 6 7 Identifying a new peak top Item Description 1 The shoulder has a maximum but the cumulati
85. meter acquisition method consists of experiments and periods In the Acquisition Method Browser pane create a sequence of acquisition periods and experiments for the instrument Users can also open a method previously created in the Tune Method Editor Experiments An experiment includes the instrument settings and the scan type during an MS scan A set of MS scans performed for a specific amount of time is called a period An acquisition method in which the MS parameters and actions are the same through the entire duration is called a single period single experiment method In looped experiments MS settings are changed on a scan by scan basis For example if the sample contains two compounds A and B users may want to loop an MS MS experiment of compound A with an MS MS experiment of compound B to obtain information about both compounds in the same run The mass spectrometer method will alternate between the two scan types Other examples of looped experiments include alternating between positive and negative modes in a run and Information Dependent Acquisition IDA methods Periods A period can contain one or more looped experiments In a multi period acquisition method experiments are performed for a specified amount of time and then switch to another set of experiments Periods are useful when the elution time of the compounds in an LC run is known The instrument can perform different experiments according to when the compounds elute to
86. n names from their preset names if required The switching valve is sometimes used to switch the flow of solvent to waste or toa different column The preset position names are A and B Inthe Change Position Names list select a position Inthe Change Position Names list rename the preset position names A and B to Inject and Divert or to Column and Waste depending on how the valve is plumbed 3 Inthe Total Time min column click a cell and then type the total time the valve will remain in this position 4 Inthe Position column click a cell and then in the Position list select the valve position Repeat the steps 3 and 4 for each switch of the valve required during acquisition Save the file Set the Diode Array Detector Parameters 1 With a method file open in the Acquisition Method Editor in the Acquisition method pane click the Diode Array Detector DAD icon The DAD Method Editor tab opens in the Acquisition Method Editor pane 2 Setthe desired acquisition parameters and save the file Set the Analog to Digital Converter Properties 1 With a method file open in the Acquisition Method Editor in the Acquisition method pane click the Analog to Digital Converter ADC icon The Analog Digital Convertor Properties tab opens in the Acquisition Method Editor pane 2 In the Sample section in the Rate pts sec field type the rate Note The interval and rate are proportional to each other When th
87. ng is frequently used Quadratic Calibration Equation The quadratic calibration equation is calculated as y a2x2 a x taQ The polynomial co efficients are calculated as a2 b2 bgo bs b3 b1 bgo b4 b3 a b5 b3 a2b4 b3 78 of 94 Quantitative Data Analysis a0 Ewy aj Zwx a2Xwx2 XEw The correlation co efficient as r Ja Ew YXw y a2x2 a x a9 Dy Where Dy XwXwy2 Xwy 2 bo Ewx Z w Xwx2 Xwx b Ewx2 EXw Xwx Xwx b2 Xwy Xw Xwxy Xwx b3 Xwx2 Xwx Xwx3 Xwx b4 EXwx Ewx Xwx wx b5 Xwxy X Xwx Xwx2y Xwx Report Templates Add the following information to report headers and footers Gj Tip Make a backup of the existing report templates before editing them Table 6 8 Basic Design Elements Table 6 9 Acquisition Elements Element Definition Printing Date The date the document was printed Printing Time The time the document was printed Operator The operator who printed the document Workstation The workstation from which the document was printed from Page n of N Indicates the page number of total number of pages Custom Field Create custom text here Analyst Version Indicates the version of the Analyst software User Type Indicates the user type Security Electronic Signature Indicates whether the electronic signature feature security is enabled or disabled Element Definition Acquisition File
88. ng scripts available Processing scripts stored in the API Instrument project appear in the Scripts menu Project Information Contains all project information and settings for the project This folder cannot be stored in a subproject Quantitation Methods Contains all quantitation methods used Quantitation methods have a qmf extension Results Contains all quantitation results table files rdb extension Templates Contains report templates rpt extension Software Security The software has a number of functions for configuring and managing security The Analyst software administrator can Choose a security mode to best suit the needs of the operating environment Add and delete users and roles e Set access rights to users and roles as required e Control access to remote instrument stations e Control access to project files For more information about Analyst software security refer to the Laboratory Director s Guide Workspaces A workspace is a particular arrangement of windows and panes including any associated file or files For example while working on a particular data set users can open and size various windows to help with the analysis This arrangement or workspace can be saved so that the next time users look at the data the window arrangement is identical In Quantitate and Explore modes users can have multiple workspaces per session This means that different workspaces can be
89. of the analyte peak Analyte Concentration The actual known concentration of the analyte this applies to standard and quality control sample types Zeroes are displayed for solvent blank and double blank sample types N A is displayed for unknowns Analyte Retention Time The chromatographic retention time as determined by the software Analyst 1 6 1 Software Advanced User Guide 90 of 94 Quantitative Data Analysis Table 6 14 Results Tables Sample Column Fields Continued Field Analyte Expected Retention Time Definition The retention time of the representative sample as taken from the quantitation method Analyte Retention Time Window The retention time window as specified in the quantitation method Analyte Centroid Location The intensity weighted average retention time for the analyte The peak areas up to and after this time are identified Analyte Start Scan The cycle number associated with the period experiment combination where the peak begins Analyte Start Time The time associated with the period experiment combination where the peak begins Analyte Stop Scan The cycle number associated with the period experiment combination where the peak ends Analyte Stop Time The time associated with the period experiment combination where the peak ends Analyte Integration Type The method by which the baseline was found and integrated when the peak was found The t
90. olling Contour Plot Colors Users can define the colors on a Contour Plot graph to provide better contrast and display data specifications according to their needs For example setting the intensity wavelength and changing the color of the values for Below Low Data and Above High Data can eliminate background noise in a Contour Plot The Below Low Data and Above High Data buttons shrink and expand on the color bar if the slider controls are moved When the contour plot colors are changed the new colors become the preset colors for all subsequent graphs Table 5 2 Right Click Menu for Contour Plot Panes Menu Function Show DAD Spectrum Opens a new pane with the DAD spectrum Extract Wavelengths Extracts up to three wavelength ranges from a DAD spectrum to Use Range display the XWC Extract Wavelengths Extracts wavelength ranges using the maximum wavelengths Use Maximum Zoom to selection Zooms in on the selected area Add User Text Adds a text box at the position of the cursor Undo Zoom Returns the graph to the original scale Delete Pane Deletes the selected pane Show Cross Hair Shows the Cross Hair nm min View a Contour Plot A Contour Plot can be viewed only after acquisition Users can view a Contour Plot from TIC XIC TWC or XWC graphs TICs and XICs are available for all wiff data files TWCs and XWCs are available only for data acquired by a DAD 1 In Explore mode open a data file as a
91. only data pertaining to the specified pattern For a spectrum the calculator removes all unmatched data For a Advanced User Guide Analyst 1 6 1 Software 35 of 94 Qualitative Data Analysis chromatogram the calculator calculates the elemental target for each of the underlying spectra and regenerates each point in the chromatogram on the basis of these new spectra Mass Property Calculator The Mass Property calculator determines various properties such as exact mass the average mass the mass accuracy and the mass defect of a mass of interest The results generated by this calculator depend upon the number of input fields completed Isotopic Distribution Calculator The Isotopic Distribution calculator determines the isotopic distribution based on an entered formula This allows users to distinguish between compounds with the same mass based on relative intensities of isotopes The calculated isotopic distribution can be displayed in graphical or text format on the Isotopic Distribution pane overlaid on the active spectrum or exported to a separate file Centroided Peaks Calculating the centroid of a peak converts peak distribution values into a single value of m z and intensity that represents the peak Centroided data collected in profile mode simplifies the data and reduces the file size Centroided data provides more accurate peak assignment and reduces the amount of data but it also removes the information about the peak shap
92. played as a function of time the time at which a particular scan was obtained which can be derived from the scan number When data is viewed as a spectrum mass specific information about a compound is obtained A chromatogram provides a general view of the data usually time dependent when using an LC column but it does provide information about the components of a peak A spectrum however looks at a particular peak and provides the molecular weight of the corresponding compound which can be used to find more specific information For example while a chromatogram may show only one peak that peak can represent more than one compound that is different masses A spectrum shows all of the masses that make up a peak including the intensity of each mass Chromatographic data can change in both time and intensity if there is a change in the chromatographic conditions in a given sample Spectral intensities may change but the masses are fixed because the mass of a compound does not change There are two ways to generate spectral data e If only one scan is acquired then the data is shown as a spectrum From a chromatogram A typical spectrum is shown with the molecular weight labeled with the m z mass to charge ratio on the x axis The intensity is shown on the y axis A chromatogram is a graphical display of the data obtained from the analysis of a sample It plots the signal intensity along an axis that shows either time or scan numbe
93. poundLib mdb v Security Information Use a specific user name and password User Name admin Password Figure 5 5 Library Manager tab 3 Inthe Available Libraries section click the alias of the database to connect to and then click Connect 4 Toallow other users to access the database select the Available to all users of this machine check box 5 Click OK Advanced User Guide Analyst 1 6 1 Software 47 of 94 Qualitative Data Analysis Connect to a Local Library Database 1 Click Tools gt Settings gt Optimization Options The Optimization Options dialog opens Click the Library Manager tab 3 In the Available Libraries section click New Add Library Library Information Enter a Name for the Library Database Information Database type MS Access local OMS SQL Server server Enter the location of the database v Browse Security Information Use a specific user name and password User Name Password Save Cancel Figure 5 6 Add Library dialog Type a name for the library In the Database Information section select MS Access local Type the database location ee ME SES In the Security Information section if required type a user name and password to access the database 8 Click Save Connect to a Server Library Database 1 Click Tools gt Settings gt Optimization Options The Optimization Options dialog opens 2 Click the Library Manag
94. r Advanced User Guide Analyst 1 6 1 Software 31 of 94 Qualitative Data Analysis The software plots intensity in counts per second cps on the y axis against time on the x axis Peaks above a set threshold are labeled automatically In the case of LC MS the chromatogram often is shown as a function of time Spectra A spectrum is the data that is obtained directly from the instrument and normally represents the number of ions detected with particular mass to charge m z values It is displayed as a graph with the m z values on the x axis and intensity cps represented on the y axis In the case of MS MS data the intensity is associated with two masses the precursor ion mass Q1 and the product ion mass or masses Q3 Background Subtraction Background subtraction reduces the amount of noise in a spectrum by subtracting either one or two ranges that contain noise from a range that contains a peak Users can move the ranges independently or lock them and move them as a single entity within the graph to optimize peak isolation or to isolate another peak Locked Background Subtract is the preset setting Perform a Background Subtraction from a Spectrum 1 Open a data file 2 Select a background range WE Xic ore MRM G pais 400 0 200 0 amu from Sample 1 AFIS012 of QuanData Wit Unknown lon Source Max 640 ops Peak of Interest intensity eps Background Time min 3 Hold down the Shi
95. race For full scan data this allows users to visualize the differences between several sample spectra If one or more panes are chosen then each XIC will appear in a separate pane N Tip To overlay fewer than four graphs in the same pane press Ctrl right click in a pane and then click Appearance Options In the Appearance Options dialog Multiple Graph Options tab select Yes for the Overlay Multiple Panes fields for Spectrum and Chromatogram Select the first pane to be overlaid Click Explore gt Overlay Click in the second pane The graphs are overlaid showing the two traces in different colors Tip To view a color coded list of the overlaid graphs right click the title _ bar of the pane wu Analyst 1 6 1 Software Advanced User Guide 38 of 94 Qualitative Data Analysis Cycle Between Overlaid Graphs 1 Select a pane that contains overlaid graphs 2 Click Explore Cycle Overlays The display changes so that the next graph in sequence is shown in the foreground Sum Overlays If two or more graphs overlaid are overlaid users can sum the graphs to get a new trace Each point on the new trace is the sum of the points from the graphs Summing several overlays of similar data type can make subsequent processing operations easier and faster For example users can overlay several XICs sum them and then smooth the summed overlay to remove noise Summing overlays is similar to generating a TIC
96. romatogram e To set noise and area thresholds it uses the resulting baseline noise This process is identical to how the preset values are set for peaks in normal methods These integration parameters are applied to all other samples Ifthe batch being examined does not contain quantitation information sample type and concentrations the retention time and thresholds are calculated separately for every peak as for fully automatic methods Metric Plots A metric plot graphically shows the data in a Results Table column plotted against the x axis or the y axis or the data in two columns plotted against each other This section describes how to generate and work with metric plots A few predefined metric plots are also included e nt Std Response to locate problem sample e Analyte Area versus Height to verify chromatography behavior PK profile conc versus time point to run after Sample query Use metric plots to plot a given column such as Analyte Peak Area Accuracy or Calculated Concentration from the Results Table Two Results Table fields can also be plotted against each other Then points that appear outside the normal range can be investigated Metric plots are often used with queries For more information about queries refer to the Help Generate metric plots in the following ways Use the Plot button to plot a column or columns of the current Results Table but not save the plotting criteria e Create a
97. s It does not however allow selecting an internal standard all ions are treated as analytes Use the Automatic Quantitation wizard after acquiring the sample in the following scenarios e Want to select calibration e Do not want to adjust peak finding and integration parameters Do not want to select analyte peak names Do not want any internal standards Do not want to review peaks during method creation or have different compounds in every sample When peaks are only being integrated they do not need to be verified as no concentration calculation is required In this case use the Automatic quantitation method creation which allows reviewing the peaks after integration has taken place Find Peaks Using an Automatic Method The software uses the standard peak detection process with the following exceptions e It uses the bunching factor and the number of smooths from the wizard as is e It calculates the expected retention time and noise and area thresholds separately for every peak Quantitation Method Editor Use this option after acquiring the sample to do the following e Adjust peak finding and integration parameters e Select analyte and internal standard peaks e Select calibration Use the Quantitation Method Editor to do three additional tasks e Sum ions for integration e Use an internal standard from a different period or experiment if the internal standard was acquired in a different period or experimen
98. senes 5 D ngfmL 3 7Oe 104 1 51e004 M amp B seres 100 ng ml 73e 004 1 Oe 004 E series 20 0 ng mL 7 81 e 004 8 040 2C3 M9 Unknown concentra 1 23e 004 8 39e 003 Unknown concentra B 71e 003 5 71e 003 23 Unknown concentra 1 12e 004 7 18e 003 25 Unknown concentra 1 32e 104 7 2604003 Unknown concentra 1 25e 004 1464 003 z Unknown concentra 1 10e 004 5 5De 003 31 Unknown concentra 1 3663004 794e4003 j Figure 6 16 Sample Summary Layout View Analyte Layout View The Analyte Layout view contains the data for a particular analyte all other analytes are hidden For example if analyte A is selected all the data for analyte A is seen The columns that are displayed depend on the columns selected in the Results Table Columns dialog and the settings selected on the second page of the Quantitation Method Wizard An Analyte Layout view with Peak 1 selected might look like Figure 6 17 If this layout is compared with the Full Layout view notice that every other row has been excluded Sample Name File Name ry oe prn Hh Pini o m Use Record um d umm B senes O blank QuanData Wit 2 45e 002 6 02e 001 0 00 E IS BE series 01 ngmL QuanData Wit 7 80e 002 2 53e4002 0 00 m mi 5 B series 0 2 ngmL QuanDats wit 1 550 003 5086 002 0 00 Z n B senes 0 5 ng mL QuanDats Wit 3 32e 003 1 04e 003 0 00 z r 3 B senes 1 0 ngfmL QuanData Wit 7 12e 003 2 33e 003 1 0n
99. stored in the following locations MS Access on a local database MS SQL Server Before using the Library Search feature determine where the library database is stored and connect the computer to that location Library databases can be stored locally and over a network Use an alias to connect to a database In this case the alias specifies a connection to a specific database and may include the user name and password required to access the database For example a user may have a small library database of identified compounds on a computer and the company may have a central database that is used occasionally by the users Creating Analyst 1 6 1 Software Advanced User Guide 46 of 94 Qualitative Data Analysis aliases for each database allows the user to switch between them quickly For information about creating aliases and connecting to databases refer to the Help Switch Between Existing Library Databases Users can connect to any databases that have aliases that are already set up 1 Click Tools gt Settings gt Optimization Options The Optimization Options dialog opens 2 Click the Library Manager tab Optimization Options Compound Database Options Library Manager Available Libraries Choose a library to connect to Available to all users of this machine Library Information Database Information Database type MS Access local OMS SOL Server server Location of database C Analyst Data Com
100. sults Table to a txt file for use in other applications such as Microsoft Excel All possible data in the table or just the data in the visible columns can be exported The data in a Results Table can be sorted in three different ways e Quickly sort the table on one to three columns using one of the Sort buttons This sort criteria cannot be saved Create a table specific sort to save the sort criteria with the current table Table specific sorts enable sorting the current table on one to three columns and saving the criterion for use with that table e Use a previously created preset sort Create and save a sort and later apply it to a Results Table Tip To save a sort or any other table setting right click in the table and e then click Table Settings gt Export To New Table Settings The sort and gt other parameters can be used in the current project To use the table settings in a different project copy it to another project Click Tools gt Project gt Copy Data A new project must be created and selected to be available for use Define the Layout of Results Tables Four predefined views of the Results Table are available e Right click in the Results Table and then click one of the following e To view the Full layout click Full All the analytes are displayed To view the Summary layout click Summary and then click a field name Analyst 1 6 1 Software Advanced User Guide 82 of 94 Quantitative Data
101. t than the analyte Edit an existing method The Semi Automatic Method Editor The Semi Automatic Quantitation Method Editor is part of the Batch Editor Use the Semi Automatic Quantitation Method Editor to select quantitation information such as sample type and Advanced User Guide Analyst 1 6 1 Software 63 of 94 Quantitative Data Analysis sample concentration prior to data acquisition This preparation makes performing subsequent quantitative analysis easier Alternatively a full method can be selected in the Batch Editor which is then automatically applied at the end of the batch run to generate the quantitation Results Table Use this option in the following scenarios Have not yet acquired any samples e Want to select names and masses for analyte and internal standard peaks e Want to select concentrations and sample types on the Quantitation tab in the Batch Editor but do not have any other quantitation method e Want to edit the quantitation method if necessary at a later time Find Peaks Using a Semi Automatic Method The software uses the standard peak detection process with the following exceptions e tuses the bunching factor from the Quantitation Method Options dialog and the number of smooths from the Create Semi Automatic Quantitation Method dialog as is tuses the most concentrated standard as the representative sample To establish a retention time it uses the largest peak within that ch
102. t the background range within the TIC and set it as background 3 Click Explore Background Subtract to File Advanced User Guide Analyst 1 6 1 Software 33 of 94 Qualitative Data Analysis Background Subtract to File xi Select a project location and file name for the processed file Click the Start Processing button to start the arithmetic operation r Dutput Project and Filename Analyst Project Example X Output Filename QuanData subtracted wif M Open the new file immediately in Analyst r Progress Start Processing Gor Processing Figure 5 1 Background Subtract to File dialog 4 n the Output Project and Filename section type the project and file names for the resulting file 5 Click Start Processing The progress bar displays the progress of the subtraction process If the Open the new file immediately in Analyst check box is selected then when the subtraction is complete the file will appear 6 Ifthe check box is cleared when the subtraction is complete click Finish Baseline Subtract Baseline subtract removes a constant or slowly varying offset from a set of data This is useful in locating small peaks that are obscured by noise The software uses the following algorithm in performing a baseline subtraction e Every data point in the data set is considered as the center of a window in mass or time with a user definable width measured in amu or minut
103. table specific plot to save the plot criteria with the current table Analyst 1 6 1 Software Advanced User Guide 64 of 94 Quantitative Data Analysis Create a global plot to save the plotting criteria for use with future Results Tables QC unknown blanks double blanks or solvents cannot be seen on the calibration curve but metric plots can be generated of them IS Peak Area vs Index minoxidol 7 4e5 7 0e5 6 0e5 5 005 4 0e5 3 0e5 E g z 3 5 o E 2 e x o a n 2 05 1 0e5 Figure 6 1 Example of a metric plot Item Description 1 Double blanks Generate a Metric Temporary Plot 1 With a Results Table open do one of the following To plot the data on the y axis with the x axis as an index click the heading of the column for the data to be plotted To plot the data from the first selected column on the x axis and the second selected column on the y axis select two columns by pressing the Ctrl key and clicking the column headings 2 Above the Results Table click the Metric Plot by Selection icon The metric plot opens 3 Right click in the plot pane and then click Data Legend to view an explanation of the colors used by the plot 4 Right click in the plot pane and then click Point Legend to view an explanation of the symbols used by the plot Generate a Metric Plot and Save the Plot Criteria 1 Open the Tutorial Results Table rdb Results Table 2 Right clic
104. te peak mAu Analyte Wavelength Ranges The range of wavelengths nm IS Peak Area for DAD The area of the internal standard peak mAU min IS Wavelength Ranges The range of wavelengths nm IS Peak Height for DAD The height of the internal standard peak mAU Calculated Concentration for DAD Table 6 17 shows the fields that can be added to the Results Table for data acquired by an ADC analog to digital converter Table 6 17 ADC Fields Field Analyte Channel Definition The ADC channel from which the analyte was acquired Analyte Wavelength Ranges The range of wavelengths nm IS Channel The ADC channel from which the internal standard was acquired IS Wavelength Ranges Advanced User Guide The range of wavelengths nm Analyst 1 6 1 Software 93 of 94 Quantitative Data Analysis Results Table Tips Table 6 18 Results Table Tips curves To do this do this Table specific queries To Right click anywhere in the Results Table and then click Query gt view the entire table again Show All The query can be applied again or edited To examine calibration Right click anywhere in the curve click Active Plot and select the curve to be plotted on top Sample statistic review To Select the Display the Data Set s check box and then in the review an individual peak Data Point column double click the data point that represents
105. tential by performing looped experiments in succession that is one compound dependent parameter followed by then next compound dependent parameter It optimizes for source dependant parameters by making an injection for each parameter Use FIA optimization to optimize both compound and source dependent parameters using LC at higher flow rates Analyst 1 6 1 Software Advanced User Guide 16 of 94 Instrument Tuning and Calibration Infusion Infusion is the continuous flow of the sample at low flow rates into the ion source using a syringe pump During the infusion optimization process the software can select precursor and product ions and optimize for declustering potential collision energy and collision cell exit potential for both The voltages of these ion path parameters are gradually increased or decreased to determine the maximum signal intensity for the precursor and product ions Use infusion optimization to optimize compound dependent parameters only at much lower flow rates than those used during LC MS analysis Advanced User Guide Analyst 1 6 1 Software 17 of 94 Instrument Tuning and Calibration AB SCIEX 18 of 94 Batches 3 A batch is a collection of information about the samples to be analyzed Samples are usually grouped into sets to make it easier to submit them Grouping the samples into a set also reduces the amount of data that must be typed manually A set can consist of a single sample or multiple sampl
106. terpretation pane click the connect button The pointer changes to the connecting tool 3 Click the spectrum graph that is to be connected to the Fragment Interpretation tool The connected graph indicator in the lower left corner contains the name of the graph connected to the Fragment Interpretation pane The connection is broken when either the graph or Fragment Interpretation is closed If the connected wiff file has more than one sample the Fragment Interpretation pane updates automatically as users scroll through the samples Advanced User Guide Analyst 1 6 1 Software 43 of 94 Qualitative Data Analysis Match Fragments with Peaks Click Explore gt Show gt Show Fragment Interpretation Tool With a mol file in the Fragment Interpretation pane select a cell in the Fragment List that is shown in bold In the spectrum the software highlights the matching spectral peak in the color selected under the Options tab In the molecular structure the bond is highlighted If a row that has more than one matching fragment is clicked the spectral peak that is closest to its monoisotopic mass is highlighted in the mass spectrum in the color specified in the Options tab Select a Bond in a Molecular Structure Click Explore gt Show gt Show Fragment Interpretation Tool With a mol file opened in the Fragment Interpretation pane click a single non cyclic bond in the molecular structure The two resulting fragments appear
107. the saved text file is not visible then in the Files of type list select Al Microsoft Text Driver txt csv Files with the extension txt appear in the P field Click Open If an autosampler is being used then the Select Autosampler dialog opens In the autosampler list select the autosampler Click OK The sample table fills with the details from the text file Set Quantitation Details in the Batch Editor Optional If users do not want select quantitation details post acquisition then Quantitation methods can be included with a batch and used to define the quantitation details prior to submitting a batch Internal Standard and Standard columns appear in the Quantitation tab according to the quantitation method selected in the Sample tab 1 DU Ov me x M With a batch file open in the Batch Editor window click the Quantitation tab Select the set In the cell select a Quant Type for all the samples If applicable type the peak concentration in the Analyte column If applicable type the Internal Standard Repeat the preceding steps for each set in the batch Save the file Advanced User Guide Analyst 1 6 1 Software 21 of 94 Batches AB SCIEX Analyst 1 6 1 Software Advanced User Guide 22 of 94 Device Methods If creating an acquisition method file from an existing file the user can use some or all of the device methods in the acquisition method Use the Acquisition Method Editor to customiz
108. thing Options x Previous Point Weight i Current Point Weight Next Point Weight fos Cancel Help Figure 5 10 Smoothing Options dialog 3 Type the weighting factor in the following fields Previous Point Weight field Current Point Weight field Next Point Weight field 4 Click OK The data set is smoothed replacing the current data set in the pane Smooth Data using Gaussian Smoothing Q Tip To undo smoothing click Edit gt Undo The software supports one level of undo 1 Select a pane containing a chromatogram or spectrum 2 Click Explore Gaussian Smooth Gaussian smooth options x Gaussian filter width Z5 of minimal 100 distance between points Limit of gaussian filter number of fi i minimal distance between points Cancel Help Figure 5 11 Gaussian smooth options dialog Advanced User Guide Analyst 1 6 1 Software 55 of 94 Qualitative Data Analysis In the Gaussian filter width field type the width used to find the weighting of neighboring points as a percentage of the distance between the two points In the Limit of gaussian filter field type the limit of the Gaussian curve given in multiples of the distance between points Click OK The data set is smoothed replacing the current data set in the pane Spectral Arithmetic Wizard The Spectral Arithmetic Wizard performs arithmetic operations using two complete chromatograms to create a new chromatogram which
109. tler smooth than the Gaussian algorithm and it takes a long time to smooth very noisy data Gaussian Smoothing Algorithm Gaussian smoothing involves replacing each data point with the weighted average of a number of data points on either side of it The weighting for each new data point is calculated on the basis of a Gaussian curve It is a coarser smooth than the smooth algorithm but it is good for smoothing very noisy data Set two values when using the Gaussian smoothing method Gaussian filter width of minimal distance between points This value shows the width used to calculate the weighting of neighboring points The width is described in terms of percentages of the distance between two points in the scan where the preset width of 10096 gives a distribution that is as wide as the distance between data points Limit of Gaussian filter number of minimal distance between points This value corresponds to the limits of the Gaussian curve shown in multiples of the distance between points For example the preset value of 10 creates a Gaussian curve that truncates after ten data point widths on either side of the center Analyst 1 6 1 Software Advanced User Guide 54 of 94 Qualitative Data Analysis Smooth Data using the Smooth Algorithm e Tip To undo smoothing click Edit gt Undo The software supports one level of undo j 1 Select a pane containing a chromatogram or spectrum 2 Click Explore Smooth Smoo
110. ulators tab of the Appearance Options dialog To open the dialog click Tools gt Settings gt Appearance Options i gt Elemental Composition Calculator The Elemental Composition calculator determines potential molecular or amino acid compositions based on a target mass to charge ratio Type this ratio manually or select it from an active spectrum This calculator creates a table with the possible element or amino acid combinations making up the mass of interest and the characteristics of each Type or select values for such parameters as tolerance electron state and number of charges Users can also type a list of possible elements and put a limit on the number of each Hypermass Calculator The Hypermass calculator determines the distribution of a multiply charged envelope based on an uncharged mass Users can select the uncharged mass including the adduct and its polarity The calculator displays a graphical representation of the Hypermass series which can be overlaid onto the active spectrum A list of the Hypermass data is also available Elemental Targeting Calculator The Elemental Targeting calculator reduces the data spectrum based on a specific pattern primarily one corresponding to isotopic distributions It can also search an MS data spectrum for a specific pattern of peaks which can be entered either as a formula or as an isotopic distribution If the calculator finds a match it creates a reduced plot containing
111. umes that all points along the curve have the same importance Select from the types of weighting in Table 6 2 Table 6 2 Types of Weighting Weighting Description 1 x To place some additional emphasis on lower value points use a weighting factor of 1 x 11x Use a weighting of 1 x to place much higher emphasis on lower value points tly Use a weighting factor of 1 y when calibrating by the area y axis rather than by the concentration x axis and some emphasis needs to be placed on lower value points A weighting of 1 y is a variant of 1 x where y and x should be proportional to each other 1ly Use a weighting factor of 1 y when calibrating by the area y axis rather than by the Concentration x axis and a much higher emphasis needs to be placed on lower value points A weighting of 1 y squared is a variant of 1 x squared where y and x should be proportional to each other In x Use the logarithm of x to place more emphasis on higher value points In y Use the logarithm of y to place more weight on higher value points Use when calibrating by the area y axis rather than by the concentration x axis Integration Algorithms The Analyst software has two integration algorithms the original Analyst Classic integration algorithm and the IntelliQuan integration algorithms The IntelliQuan algorithm provides more consistent peak finding and integrated functionality with fewer parameters that require a
112. under one or more peaks that follow the precursor These peaks are called product peaks When the software performs an exponential skim it subtracts the area below the skim from the product peaks and gives it to the precursor peak It then subtracts the small area above the skim from the precursor peak and adds it to the first product peak The software uses the following criteria to determine whether it will use exponential skimming Exponential Peak Ratio Exponential Adjusted Ratio e Exponential Valley Ratio Analyst 1 6 1 Software Advanced User Guide 74 of 94 Quantitative Data Analysis Y ps aN Figure 6 11 Separating peaks an exponential skim Item Description 1 This area is subtracted from the precursor peak and added to the first product peak Exponential skim 2 3 Cluster baseline 4 These areas are subtracted from the product peaks and added to the precursor peaks Queries A query is a method of selecting only those records that meet certain criteria Users can use queries to view particular parts of the data in the Results Table that interests them based on textual or mathematical selections When a query is used the table displays only the rows of data that meet the selected criteria All columns are displayed Selections can be further refined by running a second query on the rows displayed by the first query Use pre defined choices and typed entries to create a query that can be exec
113. uted saved or modified Each line of the query works like a Boolean search that runs against Results Table columns to determine which records to display Each line of the query selects only the records for display that meet its criteria A preset or table specific query can be defined Queries on Sample Type For a query designed to select only the standard sample type the Results Table displays only those rows of data that contain Standard in the Sample Type column Default Queries and Table Specific Queries A default query is generally used to identify samples that do not meet certain criteria A table specific query is generally used to identify records that meet certain criteria The default query is generally used to find problems with quality control or standards If the concentration and maximum variation of the QCs and standards are selected in the Quantitation Advanced User Guide Analyst 1 6 1 Software 75 of 94 Quantitative Data Analysis Method Wizard then the Results Table displays only those samples that lie outside this range If the Results Table displays nothing then the samples are all right Table specific queries are run against a displayed Results Table to select records that meet certain criteria Design these queries through the menu that is available by right clicking in the table Save and export a query to make it available for future Results Tables Table Specific or Global Settings When working with tabl
114. ve crest area is not greater than two thirds of the area threshold Analyst 1 6 1 Software Advanced User Guide 72 of 94 Quantitative Data Analysis Find the Peak End The software declares a peak end point when one of the following occurs The difference between two consecutive points fails the noise threshold test The software detects the start of a new peak In either case the lowest bunched point from the last five bunches is considered to be the actual end point of the peak In general the software finds several peaks for each chromatogram The peak it selects is the one whose retention time is the closest to the expected retention time specified in the method If no peak has a retention time within the specifications the software marks the peak as not found A Figure 6 8 Finding peaks Item Description 1 The shoulder has no separate maximum point ol Advanced User Guide Analyst 1 6 1 Software 73 of 94 Quantitative Data Analysis Figure 6 9 Finding the peak end case 1 Item Description 1 Exceeds noise threshold 2 Peak end 3 Does not exceed noise threshold ol Figure 6 10 Finding the peak end case 2 Item Description 1 Peak end Separate Peaks If a new peak begins before the current peak hits the baseline the software decides based on the following criteria whether to resolve the baseline by using exponential skims The skim passes
115. ware window below the toolbar and to the right of the Navigation bar Printing a window will print everything that is shown in that space e Pane Panes are parts of a window arranged in such a way that they do not overlap and are always fully visible For example the Method Editor window contains two panes the Browser pane and the Method Editor pane Information from each pane in the window can be printed Report Reports are structured sets of information created in the software Some reports can be directly printed such as calibration reports other information must be exported such as batches and quantitation Results Tables Workspace A workspace is a particular arrangement of windows and panes along with an associated file or files Printing a workspace involves printing each open window and pane in the current mode Preview Print and Export Reports Acquisition methods batches quantitation Results Tables and graph Results Tables can be exported as reports Other forms of information such as calculator data can be exported but cannot be customized with a report template Most areas seen on the screen can be printed Graphs can be previewed scaled or copied using the Print Preview feature An exported report is saved in a file format that is appropriate for programs such as Notepad Microsoft Word or Excel or certain LIMS Laboratory Information Management System software Export reports in the following formats
116. with the benefit of being able to choose which graphs to overlay For example if ten experiments are being viewed the TIC will add all ten experiments together If overlays are summed then users have the option of adding only nine of the ten overlaid graphs This procedure can be used if the data collected in the one experiment is just noise 1 Overlay the graphs that are to be summed 2 Click Explore Sum Overlays The overlaid graphs are added together Graphs Labels Graphs can be customized using the preset style for labels on graphs and chromatograms Users can select the fonts to use for peak and axis labels and the colors to use for the traces Users can also add axis labels and the type of label and precision for the peaks Add Captions to a Graph Use captions to label peaks of interest or significant points on the graph When a caption is placed beside a peak the caption stays with the peak when the graph is zoomed in or out Captions also stay with the original sample when users navigate between samples in a data file A caption contains one line of text with a maximum of 128 characters 1 In the spectrum right click and then click Add Caption The Add Caption dialog opens In the Caption box type the text 3 To change the size and style of the caption click Font To place the caption click OK Tip Ifthe position of the caption is not satisfactory then drag the caption toa different position The caption
117. ypes are manual automatic Baseline to Baseline Valley Exponential Skim and Exponential Child Analyte Signal to Noise The signal to noise ratio of the peak compared to the baseline Analyte Peak Width The ratio of the peak height to its width Analyte UV Range The UV range of the analyte Analyte UV Channel The UV channel of the analyte Analyte Peak Width at 50 Percent min A read only column displaying the peak width at 5096 of peak height Analyte Baseline Slope min A read only column displaying the slope of the baseline Analyte Peak Asymmetry A read only column displaying the peak asymmetry which is calculated by the following formula Peak End Time Retention Time Retention Time Peak Start Time Values near 1 0 indicate symmetric peaks values greater than 1 0 indicate tailing peaks and values less than 1 0 indicate fronting peaks Analyte Processing Alg Advanced User Guide A read only column displaying the processing algorithm used Analyst 1 6 1 Software 91 of 94 Quantitative Data Analysis Table 6 14 Results Tables Sample Column Fields Continued Field Analyte Integration Quality Sample Column Fields Definition Integration Quality Index indicates how well the peak is integrated Values closer to 1 indicate well integrated peaks and values closer to 0 indicate poorly integrated peaks This facilitates

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