Agilent G1734AA MassHunter Forensics Toxicology Dynamic MRM Database Kit Quick Start Guide
Contents
1. Name DefaultProiect Figure 10 Commonly edited options Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 21 22 8 To select which columns are displayed for editing right click in the Compound Setup table and click Show Hide Columns then mark the check boxes for the columns to display 9 For each compound record edit the information in the applicable columns 10 Save the project Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide To create a Dynamic MRM method To create a Dynamic MRM method you update your single time segment MRM method with additional retention times and retention time windows for every compound in the analysis To get the retention times for all the compounds run all the standards with your single time segment MRM method The process for 150 standards is described in Figure 11 50 standards Data File 1 Batch 1 Report 1 JU standards split into 50 standards Data File 2 Batch 2 xlsx Report 2 method three groups for all 150 50 standards Data File 3 xlsx Report 3 Figure 11 Example process for analyses that have more than 50 compounds 1 Run your standards with the method and chromatography that you created in To create an MRM method to run your own sample For best results run standards with subgroups that contain2. M Maimun 1 pm fen Maximum Concurrent MAMs 9 2 4 46 50 3 Codeine d3 30300 15500 162 45 3753 376 Pose 16317 pe mum Dwell Time idine 23000 21300 T0 z 3782 897 Positive imum De e BUD E 5 Clonidine 7 Postive Taago Minimum Cycle time 3500mr 8 Amphetanine dS 12400 5 45 923 Postive EA 7 Amphetamine d5 Mio 3270 o 4581 323 Postive zal 8 Amphetamine 3 Positive 36 50 lip o 5 TET E Options Minimum Data Point 64 dos Check Minimum Data Point True codone Positive ore de 2 Minimum Dwell Time 12 Oxycodone 31620 241 00 134 25 5 386 1217 Positive 120 465 5 13 d hydrococodone 30600 20200 768 29 6138 Postive 127 06 E Parameters T4 Hydrocodone 30020 177 00 4 667 378 Posive 14983 _ Cycle Time 15 Hydrocodone 30020 13800 758 5 6377 1 378 Postive 20819 S 00 Plot Type Concurrent MRMs 16 Metoprolol 282 5600 T36 23 10443 1 047 Postive 24650 T7 Metoprole 26820 11600 136 13 1 047 Postive 37328 78 Meprobamate 21800 15800 72 5 12614 1 Postive 18400 T8 Meprobamate 2800 5500 72 5 1263 1263 Postive 15483 20 Propranolol 11590 iz 73 ne 135 Pede 7
3. Save Batch As Close Batch Export LI but Figure23 Add Samples from the File menu b Select the acquired MRM data file from the Add Samples list or use the model data file ForensicTox Test Mix MRM d which is located in Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 35 36 the Example Data folder on the support disk then click OK See Figure 24 Select only one file Do not click Select Add Samples Batch Folder C Documents and Settings p ForensicTox Test Mix MRM d LCMS Forensic and Toxicology Test 10pg d Figure24 Add Samples dialog box 4 Create a new method a Click Method gt New gt New Method from Acquired MRM Data See Figure 25 nig Agilent MassHunter Quantitative Analysis Example Data For DMRM Method Creation 1 File Edit View Analyze Method Update Report Tools Help Nem 00 Batch Table Open New Method from Acquired Scan Data Sample 2 f Sa New Method from Acquired Scan Data with Library Search New Method using Manual Setup Acq Date Time 6 15 2009 10 55 PM Method File __ Tox Mix 1006 Method Setup Tasks Manual Setup Tasks gt Outlier Setup Tasks Advanced Tasks Figure 25 New Method from Acquired MRM Data selected Agilent G1734AA Forensics and Toxicology Dynamic MRM Data
4. This information is subject to change without notice Agilent Technologies shall not be liable for errors contained herein or for incidental or consequential damages with the furnishing performance or use of this material Agilent specifically disclaims any warranties for any implied warranties of merchantability or fitness for a particular purpose Agilent Technologies Inc 2009 2010 Printed in USA Second Edition May 2010 5990 5741EN OE Agilent Technologies
5. 248 2 Unit 1741 Unit 128 17 2 419 0 4 Positive Trazodone 1 372 2 Unit 176 Unit 159 25 2 797 0 4 Positive Trazodone m 372 2 Unit 148 Unit 159 37 2 797 0 4 Positive PCP L1 244 2 Unit 91 Unit 86 41 2876 04 Positive PCP 0 244 2 Unit 86 1 Unit 86 9 2876 04 Positive Oxazepam O 287 Unit 269 Unit 150 12 3 53 0 4 Positive Oxazepam O 287 Unit 241 Unit 150 20 3 53 0 4 Positive Nitrazepam Li 282 1 Unit 236 1 Unit 148 25 3 542 0 4 Positive 6 Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide Table 1 Dynamic MS MS transitions for LC MS Forensics and Toxicology Test Mix analytes and their chromatographic dependent settings continued Compound Name ISTD Precur MS1 Product MS2 Frag Collision Ret DeltaRet Polarity sorlon Res lon Res mentor Energy Time Time Min Nitrazepam Li 282 1 Unit 180 Unit 148 41 3 542 0 4 Positive Verapamil oO 455 3 Unit 165 Unit 158 37 3 554 0 4 Positive Verapamil m 455 3 Unit 150 Unit 158 45 3 554 0 4 Positive Methadone n 310 2 Unit 265 1 Unit 112 9 3 61 0 4 Positive Methadone 1 310 2 Unit 105 Unit 112 29 3 61 0 4 Positive Lorazepam 1 321 Unit 275 Unit 102 21 3 626 0 4 Positive Lorazepam Li 321 Unit 194 Unit 102 49 3 626 0 4 Positive Alprazolam Oo 309 1 Unit 281 Unit 179 25 3 727 0 4 Positive Alprazolam E 309 1 Unit 205 Unit 179 49 3 727 0 4 Positive Temazepam 1 301 1 Unit 2551 Unit 117 29 3 941 0 4 Positive Temazepam Li 301 1 Unit 177 Unit 117 45 3 941 0 4 Positive Proadifen 1 354 2 Uni
6. 285 0 4 Positive d Amphetamine Oo 136 1 Unit 119 1 Unit 66 5 1 296 0 4 Positive d Amphetamine Li 136 1 Unit 91 Unit 66 17 1 296 0 4 Positive MDA 0 180 1 Unit 163 Unit 61 5 1 332 04 Positive MDA 0 180 1 Unit 105 Unit 61 21 1 332 04 Positive Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide Table 1 Dynamic MS MS transitions for LC MS Forensics and Toxicology Test Mix analytes and their chromatographic dependent settings continued Compound Name ISTD Precur MS1 Product MS2 Frag Collision Ret DeltaRet Polarity sorlon Res lon Res mentor Energy Time Time Min Hydrocodone Li 300 2 Unit 199 Unit 159 29 14 0 4 Positive Hydrocodone Li 300 2 Unit 128 Unit 159 65 14 0 4 Positive methamphetamine O 150 1 Unit 119 Unit 92 5 1 45 0 4 Positive methamphetamine L1 150 1 Unit 91 Unit 92 17 1 45 0 4 Positive MDMA 0 194 1 Unit 163 Unit 97 9 1468 04 Positive MDMA 0 194 1 Unit 105 Unit 97 25 1468 04 Positive Strychnine oO 335 2 Unit 184 Unit 195 41 1 629 04 Positive Strychnine LI 335 2 Unit 156 Unit 195 53 1 629 0 4 Positive MDEA 0 208 1 Unit 163 Unit 107 9 1735 04 Positive MDEA 0 208 1 Unit 105 Unit 107 25 1735 04 Positive Heroin o 370 2 Unit 268 1 Unit 149 37 2 256 0 4 Positive Heroin 0 370 2 Unit 165 Unit 149 61 2 256 0 4 Positive Cocaine 1 304 2 Unit 182 1 Unit 138 17 2 376 0 4 Positive Cocaine 1 304 2 Unit 77 Unit 138 61 2 376 04 Positive Meperidine oO 248 2 Unit 2201 Unit 128 21 2 419 0 4 Positive Meperidine oO
7. Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 7 Save the method Database Browser Formula Polarity Species Precursor Product 21H22N202 iti 352 8 5 Psuedoephedi dAmphelamine dmphelamine Figure 7 Database Browser window To save a database that can be edited The database files that are installed with this kit are read only You can save a database to a new name so that you can edit it 1 Open the database ForensicTox DynamicMRM Database When the database is initially opened no filters are enabled the Search Text box is empty and no columns are selected All compound database records are displayed but none are selected 2 Click the Save As Database icon te Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 19 3 Type a name for the new database in the File name text box then click Save See Figure 8 Save As Save in Databases ex EE 2 CjJForensicTox DynamicMRM Database 3 Optimizer My Recent MH ForenTox DMRM dbase exe Documents Desktop My Network File name My Forensic database M Places Save as type Figure 8 Save As dialog box to save a database that can be edited To edit a user created database 1 Open the MassHunter Optimizer program 2 Click Import Export gt Import from Database The Database Browser opens 3 Click the Op
8. Quick Start Guide 4 Click Update Method to open the Dynamic MRM Update Options dialog box shown in Figure 13 22 Dynamic MRM Update Otions Select MassHunter 000 data file or Quant report folder 5 Method Options Update Retention Time True Update Retention Time Window True Add new Compound True i If No Peak Was Found for the Compound Keep old values Scale Factor of RT Window to Peak Width Cycle Time 500 E Threshold Peak Abundance Threshold 50 Retention Time Window Threshold 10 Retention Time Window Threshold Unit Percent Add new Compound Indicates whether to add compound if the compound did not exist in the method Restore Default OK Cancel Figure 13 Dynamic MRM Update Options This dialog box is used to add compounds to the method Retention times and retention time windows are obtained from the data file that is selected Select a MassHunter Triple Quad data file or Quantitative report folder Click the Browse button to find the file to use Change the options in the Dynamic MRM Update Options dialog box as needed Set all the Method Options parameters to True Set Add new Compound to True Set If No Peak Was Found for the Compound to Keep old values For all undetected peaks the retention time will be set to 0 These undetected MRM transitions will be listed at the top of the Acquisition Scan Segment table when sorted by retention time which lets you easily find them Agile
9. no more than 100 compounds per injection If you analyze between 50 and 100 compounds run a medium level calibration with a dwell time of 2 milliseconds Make sure your dwell time for all transitions gives an appropriate cycle time This criterion determines how many transitions you can put in one time segment Run with one time segment if you have no prior knowledge of retention times For peaks that are 5 seconds wide use a cycle time of 500 ms 10 points across the peak For 50 compounds with 2 transitions each use a 2 ms dwell time 5 5 ms total per transition Check that all the compounds are at medium level an adequate analysis concentration so that they are all detected in the sample run and their retention times obtained For easiest development of a dynamic MRM method all transitions in these data files must be detected Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 23 2 In the MRM method which you ran in the previous step click the MS QQQ gt Acquisition tab Note that all transitions are cleared the first time you update the method from MRM to dynamic MRM and are set to what is in the data file you select When a dynamic MRM method is updated compounds in the data file that are not in the acquisition method are added The LC conditions must be the same as those used to collect the data files you will use to create the method so that the retention times will be the same 3 Rig
10. solution 3 Prepare mobile phases A and B A 5 mM ammonium formate 0 01 formic acid in water B 0 01 formic acid in acetonitrile 4 Verify the system configuration For the analysis of the LC MS Forensics and Toxicology Test Mix load the method LCMS Forensics and ToxicologyTest Mix 01 m This method uses the HPLC system configuration as listed below Systems that deviate from this configuration may not completely comply with this method due to changes in system delay or dead volumes and appropriate adjustments will need to be made to the methodology Column 2 x 100 ZORBAX Eclipse Plus C18 1 8 um p n 959764 902 Wellplate Sampler h ALS SL model G1367D Pump Binary Pump SL Model 1312B configured with damper and mixer bypassed See To bypass mixer and damper on page 46 Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide Column Compartment Column SL Model G1316B 5 Check that your method is set up to make a 1 uL injection 6 Click Run gt Interactive Sample to do a single sample run or create a worklist to make multiple injections 7 If you do not see all the peaks after you process your data a Extend your Stop time in the method to 10 minutes b In the MS QQQ gt Acquisition tab set the Delta Ret Time to 3 minutes c Run the test mix again This will not affect your results but will show if retention times are different on your system There are a number of reasons your
11. 0900 T Cycle Time 7i Propranolol 28020 5600 12 23 1369 13 0 Posive 1277 IN Tne nma 22 Destromethorphan 27220 17090 T aj 1395 139 Postive 12983 23 Dieviromethermhan r1 272 nl 148 ani 152 74 13 981 13971 Pasitiwe 251 221 6 E amp Ea 2 3 a 8 2 1 D 8 1 2 4 5 E 8 vb n D 5 6 v 8b 5 2 23 A 5 Retention Time Figure 17 Dynamic MRM Viewer 11 Adjust the cycle time so that all criteria for minimum dwell time for the MS MS integrator and for good integration are met To use the MS MS integrator 64 data points are required in the retention time window Either increase the Delta Ret Time for the transition s with less than 64 points or decrease the cycle time As a general rule set the retention time factor based on reproducibility of the chromatography The transition table in the Dynamic MRM Viewer shows the average dwell time of each transition based on the number of overlapping transitions and the Cycle Time that appears under Parameters In Figure 17 the two compounds that are highlighted in pink indicate that the MS MS integrator will not work Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 29 The retention time window and the cycle time are set such that fewer than 64 data points will be collected When the cycle time is decreased to 350 ms as shown in Figure 18 minimum requirements for the MS MS integra
12. 1 4 Heroin 1 370 2 gt 165 0 Hydrocodone 1 30025 1990 1 Reset Sort rd _ Lorazepam 1 321 0 gt 2750 New Compound MDA 1 19015 1630 N i MDEA 1 2081 gt 1630 0 20 New Qualifier I MDMA 1 194 1 gt 163 0 5 New Calibration Level of panes 2 TIC MRM gt ForensicTox Test Mix MRM d 2 E x105 1 o Group By Time Segment 1 2 1 Expand All 08 Collapse All 06 Print Ctrl P 0 4 0 2 L Print Preview 02 04 06 08 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 42 44 46 Figure 29 Calibration Levels To selected Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 39 5 Click Select All to select all compounds in the data file See Figure 30 Copy Calibration Levels To Select Compounds Transition ISTD Flag a Figure 30 Calibration Levels To dialog box j Click OK k Click Validate then click on each error and correct it To correct an error type a value for the parameter that is missing or delete that transition from the method Typical errors are retention time cannot be zero or missing qualifier ratio Make sure the method is validated with no errors before you continue Save the method As a way to keep track of the method use the same name as the data file such as ForensicTox Test Mix MRM quantmethod xml m Click Metho
13. 327 13 d tydrococodone 30600 20200 GENGE 614 Posiive 8789 E Parameters 14 Hydrocodone 30020 171 00 158 4 6 367 1 378 Positive 10383 Cycle Time 350 18 _ Hydrocodone 30020 19900 i 29 6 amp 1379 Pose F Flot Options RENE 16 Metoprolol 26820 5600 13623 10443 1047 Postive 17150 me T7 Metoprolol 268 20 11600 138 13 1048 1 047 Postive 26075 T8 Meprobamate 21800 15800 z 5 12614 1282 Positive 12775 T8 _ Meprobamate 21300 5500 72 5 12 630 1263 Posive 10733 20 Propranolol 26020 11590 12 13 13 682 1 369 Postive 5 a aj cle Time 21 Propranolol 28020 5600 12 23 1369 1370 Postive e props 22 Dextromethorphan 27220 17090 152 4 13953 1 387 Postive 8383 2 Nevtrametharmhan r1 272 nl 148 ani 152 291 13 951 13971 Prsitive 176 RRI 6 E 4 2 3 amp 2 i E 0 8 1 Bb 3 4 5 6 6 3 wd n 2 3 14 5 6 7 2 2 23 A S Retention Time min Figure 18 List of corrected transitions However when you decrease the cycle time you effectively decrease the average dwell time for all transitions As an alternative you can increase the retention time window for the compounds that do not meet the 64 point criterion so that the dwell times of only the transitions overlapping with the extended window are decreased To see the effect of a retention time window increase you must close the viewer change the retention time window in the acquis
14. Levels to 1 Click Create Levels a You do not need to change the Units settings Method Table x i Time Segment lt All gt v gt Compound Reset Table View Level Name Prefix CUUmEE 7 Name Data File Type Level __ ForensicTox_Te ForensicTox Quante m Date Time pe EI Alprazolam 309 1 gt 281 0 MRM Target 1 0000 1 2 g ml ESL Lm E E NC meae d Amphetamine _ Diazepam Heroin Hydrocodone _ Lorazepam MDA 1 1 1 1 1 1 1 1 1 1 iE O 248252201 Target Cd methampheami 15915810 MEM Target 1 244 2 gt 86 1 Figure 28 Quantifier table with first compound selected lv R 38 Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide After the level is created right click the name of the first compound and click Copy Calibration Levels To See Figure 29 Name Vata rile Level Vate 1 ime ForensicT ox_Te ForensicTox Add Remove Columns Quantifier rmm TS Drm Restore Default Columns Dil High Conc L Cocaine 1 304 2 gt 1821 Auto Fit Columns p dAmphetamine 1 136 1 gt 91 0 Diazepam 1 2851 gt 154 0 n
15. R rit roms o cw Update Method Cut Copy Paste from Clipboard 23338 me oyde Figure 5 Import from optimizer shown in the Acquisition tab shortcut menu Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 15 The Database Browser is opened with the default database You can then select the compounds and product ions needed to import into the acquisition method of your choice 5 Open the MassHunter Forensics and Toxicology Dynamic MRM Database a In the Database Browser click File gt Open Database b From the D MassHunter Databases folder select the ForensicTox DynamicMRM Database folder Note that the name of the current database Read Only is displayed at the bottom of the Database Browser Figure 6 shows the Database Browser with the MassHunter Forensics and Toxicology Dynamic MRM Database loaded The database as it is shipped contains compound name formula the nominal monoisotopic mass of the compound the method s that were used for analyses The parameters for analysis include the precursor ion that gave the optimal signal and its associated fragmentor voltage at least two product ions if the compound did produce two significant product ions the optimized collision energy for each product ion In addition the abundance of each ion is shown so that you can determine the best quantitation and qualifier ions Alternatively you can select to display
16. abase Kit Quick Start Guide To update a Dynamic MRM method to include data files with errors Do these steps only if you are unable to successfully create a Dynamic MRM method with the use of the Update Method function directly from a data file For example if you get an error message such as that shown in Figure 20 when you update the method with a data file none of the compounds in that data file are included in the dynamic MRM method that you are creating You can use the steps in this topic to add the valid compounds from that data file A Quant cannot process selected data file or Quant result file has been deleted The missing Quant report D iMassHunterlDatalEmerging ContaminantslGroupsi 5 posnegl QuantReports PPCPs 1 4 Extras DMRM POSneg 01 Figure 20 In this topic you Manually generate a report for each data file Remove all errors in the manually generated quantitation method Update the dynamic MRM method with a Quant Report using the Update Method tool Do these steps after you run your standards for only the data files that cannot be used to automatically create the dynamic MRM method Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 33 34 Create a batch file for each data file To process multiple data files you create a separate batch file and report for each one You will use the report file instead of the data file to update your dynamic MRM method Do the step
17. ats Template Report File Name Printer Publish Format Queue Viewer OK Cancel Figure 32 Report dialog box Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide d In the D MassHunter Report Templates Quant folder click DMRM Method Gen xltx then click Open See Figure 33 Figure 33 Open dialog box with DMRM Method Gen xltx selected e In the Report dialog box specify the Printer to use the Publish Format and the Report File Name f Depending on your version of the MassHunter Quantitative Analysis program either click Queue Viewer to open the Queue Viewer and then minimize the viewer or mark the Queue Viewer check box g Click OK MassHunter can take one to two minutes to generate the report Use the Queue Viewer to check on the progress 10 Repeat this entire topic starting from the top of Create a batch file for each data file on page 34 for every group of standards that you need to process manually Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 43 44 Create final Dynamic MRM method 1 For the first data file for which Quant reports were manually generated a In the MassHunter Acquisition software right click within the Acquisition tab Figure 12 on page 24 and click Update Method b In the Dynamic MRM Update Options dialog box Figure 13 on page 25 click the Browse button and select the Quant folder insid
18. base Kit Quick Start Guide b Click the data file that you just added to the batch then click Open New Method from Acquired Data Example Data jx Test Mix MRM d MHLCMS Forensic and Toxicology Test Mix 10pg d QuantResults E IndexedDataConverter log My Computer cnu8083bmj Object name Objects of type My Network Places Figure 26 New Method from Acquired Data dialog box c In the Quantitative Analysis program from the Method Setup Tasks list click Concentration Setup See Figure 27 na Agilent MassHunter Quantitative Analysis New Method 1 File Edit View Analyze Method Update Report Tools Help JC MRM Compound Setup AC Retention Time Setup ForensicTox Te ForensicTox_Te i ISTD Setup Quantifier ss Alprazolam 1 309 1 gt 281 0 FE Qualifier Setup n S Camden Setup Concentrations Cocaine ___ 1 3042 1821 Diazepam 112851 1540 f 1 3702 1650 1 3002 gt 1990 E Globals Setup enmt Enit 1 Figure 27 Concentration Setup under Method Setup Tasks Hydrocodone Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 37 Click the name of the first compound in the Quantifier table Change Dil High Conc for the first compound to 1 Change Dil Pattern to 1 2 In the Method Table header change of
19. cMRM_Database ReadOnly Figure 6 Database Browser with MassHunter Forensics and Toxicology Dynamic MRM Database opened In Figure 6 several filters were enabled including Acquisition Method and Polarity You can also select Top 2 transitions based upon Abundance or response factors to refine the search Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 17 18 6 Import the required MRM transitions from the database a Select the required compound transitions from the MassHunter Forensics and Toxicology Dynamic MRM Database browser When MRM transitions are selected the Import and the Add to Import List buttons in the Database Browser become active b To load all selected compound transitions directly into the MassHunter Data Acquisition method click Import The Database Browser window closes and the selected transitions appear in the MS QQQ gt Acquisition tab Alternatively you can add the compound transitions step 6a to the Import List To do so click the Add to Import List button then click the Import List tab to see the compounds that you have added as shown in Figure 7 This lets you continue to search or filter for other compounds or to select another database for searching Compounds selected can be sequentially added or removed from the Import List When the list is complete click Import to copy the entries to the Scan Segment table on the MS QQQ gt Acquisition tab
20. d Exit to close the Method Table 6 Click Yes to apply the method to the batch Agilent MassHunter Quantitative Analysis x A Would you like to apply this method to the batch Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 7 Save the batch and click Analyze Batch to run the batch analysis At this point you are only interested in getting the retention times out of the analysis na Agilent MassHunter Quantitative Analysis Example Data For DMRM Method Creation File Edit View Ana ethod Hpdate Report Tools Help PUO 5 a quen Layout B A Restore Default Layout Batch Table Analyze Batch Sample 8 Sample Type lt Compound 1 Oxycodone v m LG v Se Tope level Aca DateTime DE TIT Mi 100ppb Forensiclox Test Mix MAM d Sample 8 15 2003 10 55 PM 8 Save the batch again now that the results are processed 9 Generate a report for the data file a Click Report gt Generate na Agilent MassHunter Quantitative Analysis Fv 7n e Data For DMRM Method Creation i File Edit View Analyze Method Upd E Gs Analyze Batch Batch Table Sample Sample Type All 8 Queue Viewer done 15 0 Sample gt ES T L rox Mis 100ppb Forensiclog Test Mix MAM d Sample __ 6 15 2009 10 55 PM 01000 Restore Default Layout Figure 31 Generate on the Repo
21. e of the data folder of the manual report that was generated as shown in Figure 34 The method is now updated with the transitions parameters and retention times found in the Quant report 2 Repeat step 1 for each data file for which a quant report was manually generated Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide You can always use that Quant Report folder to update methods which is a faster process than using the data file again Browse For Folder Please select MassHunter QQQ data file or Quant report Folder co_pos_oneseg_MRM_O1 d w co_pos_oneseg_MRM_02 d QuantReports B seg MRM 01 PeakChromatogram 2 PeakQualifierChromatogram PeakQualifiers PeakSpectrum SampleChromatogram TargetCompoundCalibration E Method Options Update Retention Time True Update Retention Time Window True Add new Compound True If No Peak Was Found for the Compound Keep old values Scale Factor of RT Window to Peak Width 1 Cycle Time 350 E Threshold Peak Abundance Threshold 50 Retention Time Window Threshold 10 Retention Time Window Threshold Unit Percent Update Retention Time Indicates whether to update the retention time of a new method Restore Default OK Cancel Figure 34 Navigation to Quant Report folder Select the Quant report folder of the data file to be used to update the method Agilent G1734AA Forensics and Toxicolog
22. ect data for a given MRM transition Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 13 14 02 04 05 08 1 Ta 1E 16 1E 2 22 24 26 28 3 S2 544 38 4 A2 44 46 48 N Counts vs Acauisition Time Imin Figure 4 Extracted chromatograms in Qualitative Analysis Chromatogram Results window The List Mode icon is circled in the toolbar of the Chromatogram Results window shown here 6 Observe the time retention data for each compound Look in the Integration Peak List window to see the retention time window 7 If the retention times for the test mix are not close to those given in the method continue to To create an MRM method to run your own sample on page 15 to update these methods on your system You can use the data file created with the expanded retention time window Delta Ret Time in acquisition or you can develop a new method as practice Import these compounds from the database and create a one segment MRM as described in To create an MRM method to run your own sample Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide To create an MRM method to run your own sample Before you can create a Dynamic MRM analysis method to run your own sample you need a standard MRM data acquisition method in which settings such as tabular compound names ISTD optional MRM transitions fragmentor voltages and collision energies are defined With the MassH
23. ee Agilent G1734AA MassHunter Forensics and Toxicology Dynamic MRM x Database Kit Quick Start Guide What is the MassHunter Forensics and Toxicology Dynamic MRM Database Kit 1 Kit Content 2 Where to find more information 3 Before You Begin 4 Installation 4 Required Reagents and Parts 4 Getting Started 5 To run the test mix 8 To process and interpret test mix data 10 To create an MRM method to run your own sample 15 To save a database that can be edited 19 To edit a user created database 20 To create a Dynamic MRM method 23 To update a Dynamic MRM method to include data files with errors 33 To bypass mixer and damper 46 What is the MassHunter Forensics and Toxicology Dynamic MRM Database Kit The MassHunter Forensics and Toxicology Dynamic MRM Database Kit lets you analyze up to 200 Forensics and Toxicology analytes with enhanced sensitivity all in a single LC MS analysis s Agilent Technologies The MassHunter Forensics and Toxicology Dynamic MRM Database Kit helps minimize method development time for your forensics and toxicology analysis when used with Agilent s recommended LC MS configuration and accessories It stores Multiple Reaction Monitoring MRM transitions a pair of precursor and product ions of the forensics and toxicology analytes included in the database and their optimized fragmentor and collision energy settings on an Agilent 6400 Series Triple Quadrupole LC MS instrument Method development can sim
24. en Database icon 4 Select user created database to open from the D MassHunter Databases folder Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 5 If you are prompted to set the database as the default database click Yes MassHunter Optimizer x 2 Do you want to set the database as default database Figure 9 6 In the Search Filter tab select options in the Filter Compounds and Search Compounds to select the compounds to import to Optimizer then Search 7 Click Import The Optimizer window displays the compounds that you selected MassHunter Optimizer Fie Edt View Import Export Optimization Tools kA EO e de BAD Optimizer Setup Precursor lon Selection Product lon Selection Compound Setup Show results summary User Lists User Notes References Compound Name Groups Formula Mass Strychnine Alkaloid 15 2R Ephedrine Amphetamine _ Eoee RT Window Instrument Name Morpheous Precursor Frag amp 1861 81 Compound Name Groups Formula Mass Synonyms User Lists User Notes References S S Psuedoephedr Amphetamine CIOHI5NO Phenylpropanolami 7 Aminoclonazepa
25. ht click the Scan segments table or in the gray area to the right or below it The menu shown in Figure 12 appears Sample Properties hALS SL BinPumpSL ColumnSL MS Qaa Tune file Stop time Acquisition Source Chromatogram Instrument Diagnostics istunes lune sml No limit s Pump Scan segments cf min Compound Name ISTD Precursorlon MS1Res Product lon MS2Res Dwell Fragmentor Collision Energy 26 d oxycodone r 32 um 10 134 13 r lon source Time fitering dE oxycodone 322 unit zu 10 134 23 ESI E Peak width 007 min Oxycodone 316 2 Unit Add Row 1101 134 13 Oxycodone r 316 2 Unit Delete Row 10 134 25 Time segments Sertraline r 306 1 Unt Sort _ 10 88 5 Bee Scan Polarity Div Valve Delta EMV Stored Settraline r 306 1 Unit 10 88 25 rit Postive rows 400 vw d hydococodone I 306 Unit Update Method 10 165 23 d amp hydrococodone I 306 Unit 0 166 a Codeine d3 n 303 Unit Copy 10 162 4 Codeine d3 r 303 Unit 10 162 45 141 gt Hydrocodone r 300 2 Unit Paste from Clipboard 10 158 28 Hydrocodone r 3002 Unit Fil Down 10 158 a z 988 me cycle INNEREN meat ins Fill Column ET Em E Figure 12 Acquisition tab with Update Method command highlighted 24 Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit
26. ition method and then open the viewer again Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide A dwell time of 2 ms or more is required to acquire data for dynamic MRM If both cycle time and overlapped peaks reduce the dwell time of a transition to below this value that transition is highlighted and the minimum cycle time and dwell time on the right is also highlighted Increase the cycle time to increase the minimum dwell time to correct the method problem At a minimum for good quantitative results peaks must have at least 10 data points In an example of a 3 second peak width a cycle time of 300 ms barely provides this If good quantitative results cannot be obtained because of too many overlapping peaks select a retention time delta that will give less than 64 points If you do select the general integrator in the quantitative method used to process standards and samples collected by this method The default is the MS MS integrator Good reproducible chromatography will enable a large number of compounds to be analyzed in one method using Dynamic MRM 12 Once a cycle time is determined for good integration 10 or more data points across a peak type in this value for Cycle Time in the MS QQQ gt Acquisition tab of the method editor Figure 19 shows the cycle time setting in the MS QQQ gt Acquisition tab Acquisition source chromatogram instrument Diagnostics Sean segments Col
27. lision Ret Time Delta Ret Precursor Product lon lon Energy Compound Name 1570 MS1 Res Lb MS2Res Fragmentor 322 Unit 304 Unit 134 13 5 2531 0 526 316 2 Unit 298 1 Unit 134 13 5 351367 1 21715 306 1 Unit 274 8 Unit 88 17 25038 1 727 306 Unit 202 Unit 166 29 613785 0514 Unit 199 Unit 158 23 6 377083 1 3783 296 Unit 250 Unit 390 15 22 64233 2 265 290 1 Unit 198 Unit 162 33 19 16847 1 917 285 1 Unit 193 Unit 162 33 19 28048 1 929 272 2 Unit 170 3 Unit 152 41 13 9528 1 397 gt d6 oxycodone Oxycodone Sertraline oa d6 hydrococodone Hydrocodone Diclofenac Diazepam d5 Diazepam 7373731713131 73131771 e s Dextromethorphan Dynamic is mr Figure 19 Acquisition tab with the new Cycle Time Note that the cycle time in the Dynamic MRM Update Method Options dialog box Figure 13 on page 25 is applied only the first time the method is created using the Update Method function After that the cycle time must be manually typed into the MS QQQ gt Acquisition tab Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 31 32 When you close the method viewer changes made to the cycle time in the viewer are not entered into the acquisition method 13 Save the method Agilent G1734AA Forensics and Toxicology Dynamic MRM Dat
28. namic MRM Database Kit Quick Start Guide 3 Before You Begin Installation 1 Check that the Agilent 1200 Series LC is properly installed and verified 2 On the Agilent 1200 Series Binary Pump SL check that the mixer and damper are bypassed See To bypass mixer and damper on page 46 for details 3 Check that the Agilent 6400 Series Triple Quadrupole LC MS instrument is properly installed and verified 4 Check that the following programs are properly installed MassHunter Data Acquisition B 03 01 SP1 or higher MassHunter Quantitative Analysis B 03 02 or higher MassHunter Qualitative Analysis B 03 01 or higher MassHunter Optimizer B 03 01 or higher 5 Install the MassHunter Forensics and Toxicology Dynamic MRM Database Follow the installation instruction on the front of the database installation disk 6 If you want to use the example methods that are included with this kit copy the methods from the support disk to the D MassHunter Methods folder or to a folder under the Methods folder 7 Copy the content of the Report Template folder on the support disk to the D MassHunter Report Templates Quant folder on your system This report template is used to create Dynamic MRM methods Required Reagents and Parts LC MS grade acetonitrile and water Formic acid highest purity Ammonium formate highest purity ZORBAX Rapid Resolution Eclipse Plus C18 HPLC Column 2 1 x 100 mm p n 959764 902 4 Agilent G1734AA Foren
29. nostics Scan segments CompoundName 1870 P2499 5 Res Product MS2 Res Fragmentor Add Row i diogcodone r 322 Unit 304 Unit 134 ia saa 05 Diyeodone r 3162 Uni 2883 Unit 134 Ta 351367 121715 Sertaine r 3061 urit 2748 Uri 88 s 1728038 1727 Import from optimizer T 306 Uri 202 Unit 156 2a 613785 0814 Codeine d3 r 303 Unit T8 Unit 152 rT 0 oy View Method Hifgocodane n 3002 Uri 188 Unit 158 23 6 377083 1 3783 Hydrochlorothiazide T 288 Unit 1688 Uni 134 13 0 Diclofenac r 236 Uri 250 Uri 100 15 2260203 228 Diazepam d5 r 2903 uri 188 Unit 152 33 1316847 1517 Paste from Clipboard Paste Clpboard Dynamic Parameters Cycle Time 500 ms EE Figure 16 View Method command Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide The Dynamic MRM Viewer appears It provides a powerful display to show you important details of your method See Figure 17 Dynamic MRM Viewer og Dynamic Statistics Average ISTD Precursor Product j Fragme Ret Pet 4 Ip st Command Nene FT ge Yl on S lon cron Y Os YT onines Ye Lime RS Dael Total MMs 3 M ETT Tisi
30. nt G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 25 The retention time window Delta Ret Time is scaled to the peak width found for that compound A scale factor of 2 will create a retention time window that is 2 times the peak width baseline to baseline Choose a larger factor if you want to acquire more data points for the transition The respective dwell times for MRM transitions will also decrease depending on the number of overlapping peaks and their respective peak widths See Figure 14 Sample Properties hALS SL BinPump SL Column SL MS QQQ r Tune file r Stop time Acquisition Source Chromatogram Instrument Diagnostics atunes tune xml No limit amp s Pump Scan segments min P Product Collision Ret Time Delta Ret B on recursor roduc collision etTime Delta Ret ef Compound Name ISTD lon MS1 Res ica MS2 Res Fragmentor Eneray hac lon source Time filtering gt ESI V Peak width 004 Acephate m 184 Unit 125 Urit 80 10 0 7735667 0 4048 Alrazine 216 Unit 132 Unit 120 20 759355 0 76 Time segments 5 Aznphosmethy 318 Unit 132 Unit 80 10 9 307384 0931 Erie eee REORUM Ce he Dees ied Carbofuran n 222 uni vau 120 15 7 083567 0708 1 O Dynamic P sive Toms 200 iv Diazinon r 305 Unit mau 180 20 1187535 1188 Dimethoate 230 Unit ium a
31. o 10 452365 0453 Malathion o 331 Unit ajunt 80 10 1050347 1 051 Metosulam n 418 Unit 175 Unit 120 25 7 530317 0 754 4 k Dynamic MRM Parameters cycles s ms cycle Cycle Time 500 ms Figure 14 If you manually select Dynamic MRM in the Scan Type under Time Segments as shown in Figure 14 before you update the method the transition table is cleared to contain only Compound1 When you update the method the compounds in the data file are added As shown in Figure 14 you can delete the extraneous Compound1 To delete the first row select the row right click the table and click Delete Row 26 Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide When you choose a data file whether collected in MRM mode or Dynamic MRM mode and update the method the scan type in the method is converted to Dynamic MRM and the compounds in the data file added to the method Sample Properties hALS SL BinPump SL Column SL MS QQQ Tune file r Stop time Acquisition Source Chromatogram Instrument Diagnostics atunes tune xml No limit amp s Pump Scan segments c min Browse ef Compound Name ISTD AG MS1 Res MS2 Res Fragmentor eo poses r lon source Time filtering gt ESI V Peak width 0 04 min Acephate 184 Unit 125 Unit 80 10 0 7735667 0 4048 Atrazine 216 Unit 132 Uni
32. or select Response Factors for MRM transitions in the Database Browser Note that the only way to add edit or update database content is to save the information in a MassHunter Optimizer project If you want to change the order of the columns drag a column heading to the desired location Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide You can right click anywhere within the compound table to get a menu of additional options You can Show Hide columns useful for advanced Search Filter operations M Search Compounds Search Text du 7 aminonitrazepam Optimized Compounds Acetominophen alpha Hydroxyalprazolam Date From 2 9 2010 v To 2 9 2010 alpha Hydroxyethylflurazepam _ alpha Hydrosytriazolam WIprazolam Select Columns Project Name Compound Name Formula Mw Groups CAS Chemical Classes Synonyms Group Name Anthelmintic Project Name MassHunter_Pesticides_Database_Project ly OOOOOORO s Method 300 Pesticides 1280LC DMRM m Nordiazepam nor Fentanyl Polarity Positive User Lists Show All Records Select Top transitions Use Abundance Response Factor Polarity Precursor Product Abundance Acq Method Project Name 166 1 ForensicTox_120 DefaultProject DefaultProject DefaultProject DefaultProject DefaultProject Current Database D MassHunter Databases ForensicT ox_Dynami
33. ply be done by importing target compounds from the database to the MassHunter Data Acquisition program The Dynamic MRM feature of Agilent Triple Quadrupole instruments provides adaptive MRM data collection methodology that requires the instrument collect data only at a predetermined time window the retention time range for the target compound for a given MRM transition More compounds MRMs can be analyzed in a single run through the Dynamic MRM feature without losing data quality See the technical note on Dynamic MRM p n 5990 3595EN for more information Kit Content Agilent G1734AA MassHunter Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide The Quick Start Guide provides an overview of the MassHunter Forensics and Toxicology Dynamic MRM Database Kit how to use it and where to find further information A copy of the Test Mix Report Example is also included on the support disk MassHunter Forensics and Toxicology Dynamic MRM Database Included in the kit is a disk that contains MassHunter Forensics and Toxicology Dynamic MRM Database B 03 00 along with related software license agreements See Installation on page 4 for a list of software requirements MassHunter Forensics and Toxicology Dynamic MRM Database Kit Support Disk The content of the disk is The Triple Quadrupole LC MS Dynamic MRM method LCMS Forensics and ToxicologyTest Mix 01 m A sample chromatogram and Dynamic MRM report obtained with the te
34. retention times can change from those determined by Agilent such as different instrument delay volume dead volumes or configuration Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 10 To process and interpret test mix data In this step you process the data file that you created when you ran the test mix The figures in this task are based on the example data file LCMS Forensic and Toxicology Test Mix 10pg d found in the Example Data folder on the support disk Your results may differ slightly 1 Open the MassHunter Qualitative Analysis program Click Cancel if you are asked to open a data file 2 Load default m method 3 Click File gt Open Data File and open the data file that you created when you ran the test mix You can also use example data file LCMS Forensic and Toxicology Test Mix 10pg d in the Example Data folder on the support disk Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide See Figure 1 02 04 06 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 Counts vs Acquisition Time min Figure 1 Example LC MS Forensics and Toxicology Test Mix Total lon Chromatogram Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 11 4 In the Data Navigator window right click TIC MRM and then click Extract Chromatograms from the shortcu
35. rt menu b Under Report Folder select the folder where you want to save this report Use the default data folder See Figure 32 Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 41 c Depending on your version of the MassHunter Quantitative Analysis program either click Add under Reports or click the Browse button next to Template file Report results file Report graphics file Generate report results file Choose a batch file from which the report results file is generated Batch folder C MassHunter Data TestMix Batch file test batch bin Graphics files uli Tum on off generating graphics files Turn off when you know you will choose report templates that do not use graphics files Turning off will reduce report generation time and disk usage Generate graphics files Load graphics settings fle Instrument Type determining numeric formats in graphics files aoa v Use existing report results file report graphics files Report folder Choose or enter the report folder When you chose to generate report results file and graphics files this folder is the destination folder When you chose to use existing report results file this folder must contain report results file and graphics files Report folder C MassHunter Data TestMin QuartReports test 1 Reports EA Choose report templates report file names printers and publish form
36. s in this task for each data file that you need to process 1 Open the MassHunter Quantitative Analysis program 2 Create a new batch in the folder that contains the MRM data you collected a Click File gt New Batch See Figure 21 ti Agilent MassHunter Quantitative Analysis i File Edit View Analyze Method Update Report Tools Help i 3 New Batch Ctrl N luit Layout Open Batch Ctri 0 j lel s e Export Figure 21 New Batch from the File menu Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide b Type a File name for the batch then click Open See Figure 22 Example Data Oo E fly ForensicTox_Test_Mix_MRM d Batch File Data Ve MHLCMS_Forensic and Toxicology Test Mix 10pg d J Test Batch 01 O QuantResults My Computer pem cnu8083bmj Filename For DMRM_MethodCreation Files of type Batch Files batch bin My Network Figure 22 Batch dialog box 3 Load the single time segment MRM data of your first standard that failed with the Update Method function a Click File Add Samples See Figure 23 na Agilent MassHunter Quantitative Analysis Example Data For DMRM Method Creation 2 File Edit View Analyze Method Update Report Tools Help i New Batch A 7 Restore Default Layout B 25 Open Batch Save Batch Te isto Time Segme
37. sics and Toxicology Dynamic MRM Database Kit Quick Start Guide Getting Started The sample data files provided in the support disk were acquired with the test mix on a system with the LC MS system configured as described in Installation on page 4 Along with the sample data files are the Dynamic MRM methods with which these data files were acquired If you review the acquisition method and sample data you will get an idea of the data acquisition data processing and result interpretation from using the MassHunter Forensics and Toxicology Dynamic MRM Database Kit To review the Acquisition Method use the MassHunter Data Acquisition program to load the method file LCMS Forensics and ToxicologyTest Mix 01 m The following data acquisition settings for the positive ion compounds are listed Acquisition method info Triple Quadrupole LC MS settings see Table 1 Wellplate sampler settings Binary pump settings Thermostatted column compartment settings Table1 Dynamic MS MS transitions for LC MS Forensics and Toxicology Test Mix analytes and their chromatographic dependent settings Compound Name ISTD Precur MS1 Product MS2 Frag Collision Ret DeltaRet Polarity sorlon Res lon Res mentor Energy Time Time Min Codeine 0 300 2 Unit 165 1 Unit 158 45 1 11 0 4 Positive Codeine o 300 2 Unit 58 1 Unit 158 29 1 11 0 4 Positive Oxycodone E 316 2 Unit 298 1 Unit 143 17 1 285 0 4 Positive Oxycodone 1 316 2 Unit 2561 Unit 143 25 1
38. st mix Acquisition methods Report templates for creating Dynamic MRM method Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide An Application Kit for the Screening of Samples for Analytes of Forensic and Toxicological Interest using LC QQQ MS MS with a Dynamic MRM Transition Database Application Note Multi Residue Pesticide Analysis with Dynamic Multiple Reaction Monitoring and Triple Quadrupole LC MS MS Application Note New Dynamic MRM Mode Improves Data Quality and Triple Quad Quantification in Complex Analyses Technical Overview Pesticide Dynamic MRM Compound Database for Screening and Identification Using the Agilent LC MS Triple Quadrupole Systems Technical Note Agilent G1734AA MassHunter Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide PDF format ZORBAX Rapid Resolution Eclipse Plus C18 HPLC Column p n 959764 902 2 1mm x 100 1 8 um LC MS Forensics and Toxicology Test Mix p n 5190 0470 4 ampoules containing a Forensics and Toxicology LC MS test mix of 25 components Where to find more information Application Notes and Publications You can find information about the MassHunter Forensics and Toxicology Dynamic MRM Database for forensic and toxicology analysis in the application notes and publications included on the support disk For more information on Agilent products go to http www chem agilent com Agilent G1734AA Forensics and Toxicology Dy
39. t 120 20 759355 0 76 Time segments ei Azinphos methyl r 318 unit 132 Unit 80 10 9 307384 0 931 8 mS ScanType Polarity Div Valve Deka EMV Stored Carbofuran r 222 Unit vau 120 15 7 083567 0709 1 0 Dynamic MRM P sive Toms 200 i Diazinon 305 Unit 153 Unit 160 20 11 87535 1188 Dimethoate Fr 230 Unit ao 10 452365 0453 Malathion 331 Unit ajunt 80 10 1050347 1 051 Metosulam 418 Unit 175 Unit 120 25 7530317 0 754 al Dynamic MRM Parameters cycles s ms cycle Cycle Time 500 ms Figure 15 7 Click OK 8 Repeat step 3 through step 6 until all the data files that contain the standards that will be used in the one Dynamic MRM method have been added Make sure nothing is changed in the method and that all the Method Options parameters in the Dynamic MRM Update Options dialog box Figure 13 are set to True and If No Peak Was Found for the Compound is set to Keep old values 9 Save the method with an appropriate name Note that all transitions must be detected in each data file used or the MassHunter Quantitative Analysis program will generate an error when you update the method Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 27 28 10 In the Acquisition tab right click the Scan segments table or the gray area next to it and click View Method See Figure 16 Acquisition source Chromatogram Instrument Diag
40. t 167 Unit 153 29 4 088 04 Positive Proadifen 1 354 2 Unit 91 1 Unit 153 45 4 088 04 Positive Diazepam 1 285 1 Unit 193 Unit 169 45 4 268 0 4 Positive Diazepam Li 285 1 Unit 154 Unit 169 25 4 268 0 4 Positive THC 0 315 2 Unit 193 2 Unit 150 20 527 04 Positive THC 0 315 2 Unit 123 3 Unit 150 30 527 04 Positive In addition to standard MRM parameters the retention time and retention window settings are listed for each compound This allows longer dwell times better signal stability and higher data quality compared to a traditional MRM method Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 7 To run the test mix Run the LC MS Forensics and Toxicology Test Mix to get a better idea of how the MassHunter Forensics and Toxicology Dynamic MRM Database Kit will work for you 1 Do a check tune to verify that the instrument operates properly Change to the Tune context in the MassHunter Data Acquisition program click Checktune to verify the instrument properly tuned Do an Autotune if Checktune reports any failure 2 Prepare the LC MS Forensics and Toxicology Test Mix The concentration of the test mix stock solution is 1 ug mL 1 ppm for all 25 components a Dilute 100 uL of the stock solution to 10 0 mL with methanol to create the final solution concentration b Transfer 1 mL of the final sample solution to a standard 2 mL sample vial for analysis The final solution is a 10 ng mL 10 ppb working
41. t menu E Agilent MassHunter Qualitative Analysis Forensic Tox Test Mix DA Method m LCMS Forensic and Toxicology Test Mix 10pq d 5 v User Chromatograms Use Highlighted Chromatograms Integrate Chromatogram Integrate and Extract Peak Spectra Smooth Chromatogram Calculate Signal to Noise Set Anchor Y Clear Results _ Delete Integrate MS 065 Intearate UVI 06 Figure 2 Extract Chromatograms on the TIC MRM shortcut menu 5 In the Extract Chromatograms dialog box a For Type select MRM b Set Transition to All See Figure 3 c Mark the Integrate when extracted check box Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide d Click OK Extract Chromatograms List of opened data files LCMS_Forensic and Toxicology Test MRM v Integrate when extracted MS Chromatogram Advanced Excluded Masses MS levet MS MS Polarity Positive NE Scans Multiple reaction monitor Transition Jan Figure 3 Extract Chromatograms dialog box After the chromatograms are extracted and integrated they are displayed on the Chromatogram Results window as shown in Figure 4 if the view is in List Mode Note the segmented chromatograms the time ranges are the predetermined time windows for the system to coll
42. tor are met When you change the cycle time in the viewer you immediately see its effects on the dwell times Dynamic MRM Viewer ISTD Precursor Product Ret ha Average Deni MOM Statistice D Y CompeundNane Presser Posty Fagre v fine Y me Y Pus Y Y za Atenolol 26720 18000 212 657 Positive 171 80 Minimum Concurent MAMs T 2 Atenolol O 267 20 14500 T4 z 215 5 Positive 17 50 Maximum Concurent MRMs z 3 Codened3 30300 16500 T amp 45 3758 376 Posive 1137 Mirimum Dwel Time SOS 7 Dwell time 350 00 ms 5 Clonidine 230 00 25 3810 897 Positive S275 rinm Gre tine d 6 AmphetaninescS Taito 12400 s 45 323 Poslive 6553 7 Amphetanine dS MT 39270 n 458 323 Posiive 638 i p 8 Amphetamine 136 10 11800 T 5 4605 1286 Positive 5650 2s a 3 Amphetamine 73610 9100 7T 13 4613 1288 Positive 6325 E Options 10 d amp oxycodone 32200 30400 134 13 5 253 526 Positive 68 44 Poin E Ti Oxycodone 31620 23810 TM 5351 1217 Postive 81 08 Mr eT T2 Oxycodone 31620 24100 T3 5 538 1217 Postive 8
43. unter Forensics and Toxicology Dynamic MRM Database Kit you can easily import all of these settings from the database to create an MRM method 1 In the MassHunter Data Acquisition program click the MS QQQ tab 2 Click the Acquisition tab 3 In the MS QQQ tab make sure the Scan Type is set to MRM not Dynamic MRM If you select Dynamic MRM instead retention times from the database are imported and will overwrite your existing conditions Refer to the technical note on the Pesticide Dynamic MRM Database on the support disk for more information on the benefits of the creation of a single time segment MRM method by use of the MassHunter Pesticide Dynamic MRM Database The concepts in this technical note also apply to the MassHunter Forensics and Toxicology Dynamic MRM Database 4 Right click an empty area on the Acquisition tab then click Import from optimizer in the shortcut menu See Figure 5 Tune file Stop time Acquisition Source Chromatogram Instrument Diagnostics atunes tune xml No limit As Pump Scan segments cf min Browse amp Compound Name 15702 PPM v Mst Product 82 Dwell Fragmento ENO Polarity lon source Time filtering gt Compoundt 200junt 200 135 0 Positive ESI zv v Peak width 007 Add Row Delatore Time segments Sort Start Delta Della e Fits ScanType DivValve chn t ewe Stored SERO DE
44. y Dynamic MRM Database Kit Quick Start Guide 45 To bypass mixer and damper The Binary Pump SL is delivered in standard configuration damper and mixer connected This step shows how to bypass the damper and mixer and convert the pump to low delay volume mode Configurations where only the damper or the mixer is disconnected while the other part is still in line are not supported by Agilent Technologies Tools required Wrench 1 4 inch x 5 16 inch p n 8710 0510 e Wrench open end 14 mm p n 8710 1924 Hex Driver 1 4 inch slitted p n 5023 0240 Preparations for Flush the system water if buffers were used otherwise IPA this procedure Turn the flow off 1 Remove the front cover by pressing the clip fasteneron 2 Use the 1 4 inch hex driver to remove fitting B from port 2 both sides of the cover of the pressure sensor 46 Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 3 Fold capillary end B away It remains unconnected Disconnect fitting A from outlet 1 of the mixer 4 Connect fitting A to port 2 of the pressure sensor Seal port 1 of the mixer with a plastic blank nut Agilent G1734AA Forensics and Toxicology Dynamic MRM Database Kit Quick Start Guide 47 www agilent com In This Guide This Quick Start Guide describes how to use the MassHunter Forensics and Toxicology Dynamic MRM Database Kit
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