Agilent MassHunter Workstation Data Acquisition for 6400 Series Triple Quadrupole LC/MS Familiarization Guide

Transcription

1 Agilent MssHunter Worksttion Dt Aquisition for 6400 Series Triple Qudrupole LC/MS Fmiliriztion Guide Before you egin 3 Prepre your system 3 Prepre to quire dt 4 Exerise 1 Develop n quisition method 6 Tsk 1. Enter quisition prmeters nd quire dt 6 Tsk 2. Determine preursor ion msses 11 Tsk 3. Find optimum frgmentor voltge for mximum response 14 Tsk 4. Determine produt ion msses 24 Tsk 5. Find optimum ollision energy for MRM quisition 30 Exerise 2 Develop Dynmi MRM quisition method from n MRM quisition dt file or n MRM method 33 Tsk 1. Crete th file from n existing MRM dt file 33 Tsk 2. Print report in the Quntittive Anlysis progrm 36 Tsk 3. Crete Dynmi MRM method using Updte dmrm 37 Tsk 4. Crete Dynmi MRM method from n MRM method 39 Exerise 3 Crete Triggered Dynmi MRM quisition method 40 Tsk 1. Crete Triggered Dynmi MRM method from Dynmi MRM method mnully 40 Tsk 2. Add/Modify ompounds in n existing dtse 42 Tsk 3. Crete Triggered Dynmi MRM method from n existing dtse 52 Exerise 4 Optimize Aquisition prmeters 56 Tsk 1. Use the Optimizer Softwre to optimize quisition prmeters 56 Tsk 2. Use the Soure nd ifunnel Optimizer progrm to optimize quisition prmeters 63

2 Use the exerises in this guide to lern how to use the Agilent 6400 Series Triple Qud LC/MS. You n do these exerises with the demo dt files, SulfDrugs, shipped with the system (in the Dt folder of your Qulittive Anlysis instlltion disk), or with dt you quire. In Exerise 1, you lern how to determine the est quisition settings for nlyzing your ompounds of interest. These instrutions help you understnd not only how to set up worklist to optimize instrument prmeters for est sensitivity in quisition, ut lso how to use the Qulittive Anlysis progrm to identify prmeter vlues produing optimum signl response. You n lso lern out the Qulittive Anlysis progrm y using the Qulittive Anlysis Fmiliriztion Guide or the Qulittive Anlysis online Help. In Exerise 2, you lern how to use either n quired dt file or the Quntittive Anlysis report results to updte dynmi MRM method. This method llows you to esily set up dynmi MRM method. In Exerise 3, you lern how to rete triggered dynmi MRM method. NOTE In Exerise 4, you lern how to use two progrms to optimize prmeters. The Optimizer Softwre helps you optimize quisition prmeters. Speifilly, it utomtes the seletion of the est preursor ion nd the frgmentor voltge for the most undnt preursor ion, seletion of the est produt ions, nd optimiztion of ollision energy vlues for eh trnsition for list of ompounds you speify. The Soure nd ifunnel Optimizer progrm helps you to find the optiml soure nd ifunnel prmeters. See the Conepts Guide to lern more out how the triple qudrupole mss spetrometer works nd why the frgmentor nd ollision energy voltges re importnt. For kground informtion, see Chpter 3, Agilent Triple Qud MS nd Sensitivity, in the Conepts Guide. See the online Help for detiled informtion on how the progrm works. Eh tsk is presented in tle with three olumns: Steps Use these generl instrutions to proeed on your own to explore the progrm. Detiled Instrutions Use these if you need help or prefer to use step-y-step lerning proess. Comments Red these to lern tips nd dditionl informtion out eh step in the exerise. 2 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

3 Before you egin Prepre your system Before you egin Before you egin, you need to hek tht your system is redy. If you pln to quire dt, you lso need to set up the instrument. Prepre your system NOTE 1 Chek tht: The Dt Aquisition progrm hs een instlled. The LC modules nd the 6400 Series Triple Qud LC/MS hve een onfigured. The performne hs een verified. The system hs een turned on. If these tions hve not yet een done, see the Instlltion Guide for your instrument. 2 Copy the dt files to your PC. Copy the folder nmed SulfDrugs in the Dt folder on your Qulittive Anlysis instlltion disk to ny lotion on your hrd disk. This folder ontins ll the dt files needed for this exerise. Do not re-use the sulf drug dt files lredy on your system unless you know tht you opied them from the originls on the disk nd you re the only one using them. Dt files tht re lredy on the system my ontin proessed results, leding to different ehvior during the exerises in this guide. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 3

4 Before you egin Prepre to quire dt Prepre to quire dt If you do not intend to quire dt ut wnt to lern how to use the Qulittive Anlysis progrm for method development, you n skip this step, whih tells you how to prepre the demo smple. You then do those tsks tht show you how to use the Qulittive Anlysis progrm with the sulf drug dt files shipped with the system. Prts List The exerise in this guide uses this equipment nd mterils: Agilent 1200, 1260 Infinity or 1290 Infinity LC modules: well-plte smpler, inry pump, thermosttted olumn omprtment, DAD Zorx olumn (see Tle 1 on pge 4) A 1 ng/µl onentrtion of the sulf mix smple (prepred in this step) Tle 1 Zorx olumns Triple Qudrupole Column Desription Film Thikness Pore Size Prt Numer 6410B, 6420, 6430, 6460 nd 6490 SB-C18 2.1mm x 50mm 1.8 µm 80Å Prepre the LC solvent. In 1-liter reservoirs of HPLC-grde wter nd etonitrile (ACN), dd 1 ml of 5M mmonium formte eh to mke 5mM mmonium formte in wter nd ACN nd use for the A nd B hnnels, respetively. 2 Prepre the smple. Add 10 µl of the sulf mix from one of the mpoules (500 µl) to 990 µl of solvent A in 2 ml glss smple vil so tht the finl onentrtion is 1 ng/µl. Cp the vil nd ple in smple lotion in the utosmpler. 3 Set up the LC olumn. Use the Agilent olumn from Tle 1. Other olumns nd instrument prmeters my e used in these exerises, ut some prmeters my need djustment, nd the results will differ. 4 Set the olumn temperture to 60 ο C. Lower tempertures my e used; however, the retention times will e longer, nd the pump pressure my exeed the limit of some LC systems. 4 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

5 Before you egin Prepre to quire dt The Eletrospry LC Demo Smple (P/N ) ontins five mpoules with 100 ng/µl eh of sulfmethizole (M+H) + = 271, sulfmethzine (M+H) + = 279, sulfhloropyridzine (M+H) + = 285, nd sulfdimethoxine (M+H) + = 311. Sulfmethizole Sulfmethzine Sulfhloropyridzine Sulfdimethoxine NOTE Determining optiml prmeter vlues for quiring smple ompound dt requires tht the Agilent Triple Qud instrument lredy e tuned on the Tuning Mix lirnt ions. Before proeeding with this exerise, mke sure you hve used Chektune or Autotune to verify tht lirnt ions eh hve the proper mss ssignment, pek width, nd signl intensity. See the Quik Strt Guide, Instlltion Guide or online Help for instrutions on tuning the instrument. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 5

6 Exerise 1 Develop n quisition method Tsk 1. Enter quisition prmeters nd quire dt Exerise 1 Develop n quisition method For this exerise you nlyze mixture of four sulfonmide ompounds. Tsk 1. Enter quisition prmeters nd quire dt In this exerise, you enter the onditions for the nlysis of the sulf drug mix. l 1 Enter LC prmeters pproprite for sulf drug mix. See Tle 2. Doule-lik the Dt Aquisition ion. Mke sure tht Aquisition ppers s the seletion in the Context text ox. If Tune is the seletion, lik Aquisition from the Context dropdown menu in the Como r. Enter the LC prmeters listed in the Tle 2. The Dt Aquisition window ppers. See Figure 1. Tle 2 Prmeter LC prmeters for sulf drug mix LC Prmeter PUMP Flowrte 800 µl/min Solvent A 5 mm mmonium formte in wter Solvent B 5 mm mmonium formte in 90:10 etonitrile:wter Grdient (min - %B) 0 min - 13% 1.80 min - 60% 2 min - 60% Stop Time 2.5 min Post Time 3.0 min INJECTOR Inj. Vol. 2.0 µl 6 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

7 Exerise 1 Develop n quisition method Tsk 1. Enter quisition prmeters nd quire dt Tle 2 Prmeter LC prmeters for sulf drug mix (ontinued) LC Prmeter Injetion Stndrd Drw Position 0.0 mm UV DETECTOR Ch A 254 nm (4 nm BW on DAD) REF A (DAD only) 400 nm (80 nm BW) COL THERM Temp 60 C for the 6460 nd 6490 with Agilent Jet Strem Tehnology 40 C for other instruments Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 7

8 Exerise 1 Develop n quisition method Tsk 1. Enter quisition prmeters nd quire dt Figure 1 Agilent MssHunter Worksttion Softwre Dt Aquisition window 2 Enter MS prmeters pproprite for sulf drug mix nd sve the method s iiims2sntest.m, where iii re your initils. See Tle 3. d Clik the QQQ t in the Method Editor window. Selet MS2Sn from the Sn Type list in the Time Segments tle. Enter the other MS prmeters s listed in Tle 3. These prmeters re in either the Aquisition or the Soure ts. Sve the method s iiims2sntest.m, where iii re your initils. 8 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

9 Exerise 1 Develop n quisition method Tsk 1. Enter quisition prmeters nd quire dt Tle 3 MS prmeters for sulf drug mix Prmeter Vlue (ESI) Vlue (AJS ESI) Inlet ESI (positive polrity) AJS ESI (positive polrity) Sn Type MS2Sn MS2Sn Delt EMV pos 400 V 200 V Mss Rnge 100 to to 400 Cell Aelertion Voltge 7 V 7 V Gs Temp 350 C 250 C for Agilent 6490 Gs Flow 12 L/min 14 L/min for Agilent C 250 C for Agilent L/min 14 L/min for Agilent 6490 Neulizer 50 psi 35 psi Sheth Gs Temperture not pplile 400 C Sheth Gs Flow not pplile 12 L/min Nozzle Voltge not pplile 0 V Cpillry Voltge positive 4000 V 4000 V Frgmentor 100 V (not djustle on 6490, omes from the Tune file) 100 V (not djustle on 6490, omes from the Tune file) If you hve n Agilent 6490, you nnot edit the Frgmentor olumn. The vlue for the Frgmentor for 6490 QQQ omes from the tune file, nd for the 6490 it is typilly loser to 380 V. Figure 2 Selet Sn Type of MS2 Sn in the QQQ t Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 9

10 Exerise 1 Develop n quisition method Tsk 1. Enter quisition prmeters nd quire dt 3 Aquire dt (optionl). Set up one-line worklist with the method you just reted. Nme the dt file iiisulfmix01.d, where iii re your initils. Designte diretory pth to hold your dt files nd method. d e f g h i If neessry, lik View > Worklist to disply the Worklist window. Clik Worklist > Worklist Run Prmeters. Verify tht the prmeters re set properly. Clik OK. Clik Worklist > Add Multiple Smples. Type iiisulfmix01.d s the dt file nme Selet iiims2sntest.m s the method nme. Clik the Smple Position t. Selet the Autosmpler, Well-plte or Vil Try. In the grphi, selet single position. Clik OK. In the Worklist window, mrk the hek ox to the left of the smple. The Worklist window is ted with the Method Editor window y defult. Clik the Worklist t to show the Worklist window. The Numer of smples is set to 1. You hve just quired full sn MS dt file to see wht ions re eing formed from the smple. This step is optionl euse you n perform the next step with n exmple dt file tht omes with the progrm. If you prefer, you n rete your own dt file s desried in this step. j Clik the Strt Worklist Run ion in the min toolr, the Run Worklist ion in the Worklist toolr or lik the Worklist > Run ommnd. 10 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

11 Exerise 1 Develop n quisition method Tsk 2. Determine preursor ion msses Tsk 2. Determine preursor ion msses In this exerise, you determine the preursor ions for eh of the sulf drugs in the quired dt file. 1 Open the quired dt file. In the Qulittive Anlysis progrm, open either the exmple file, sulfmix01.d, or the dt file you reted in Tsk 1. Enter quisition prmeters nd quire dt on pge 6. Doule-lik the Qulittive Anlysis ion. The progrm displys the Open Dt File dilog ox. When you open the sulf drug diretory fter instlltion, the Lod result dt (lower left orner) hek ox is gryed out. If you see the hek ox mrked, this mens tht the dt file(s) lredy ontins results. Cler this hek ox efore opening the file. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 11

12 Exerise 1 Develop n quisition method Tsk 2. Determine preursor ion msses Do one of the following: Selet the exmple dt file sulfmix01.d, nd lik Open. Selet the dt file you reted in Tsk 1. Enter quisition prmeters nd quire dt on pge 6, nd lik Open. By defult, the system displys the Totl Ion Chromtogrm (TIC). The figure elow shows the defult lyout. The Qulittive Anlysis progrm displys newly opened dt file with the sme lyout nd disply settings used for the previous dt file. Therefore, you MUST mke sure to return to the defult settings for this exerise. Before you egin, mke sure tht ll previous settings re returned to their defult vlues: Restore defult lyouts Clik Configurtion > Window Lyouts > Restore Defult Lyout. Mke sure the method is defult.m. (see title r) Clik Method > Open. Selet defult.m, nd lik Open. Return disply options to defult settings. In the Configurtion menu, lik eh of the Disply Options ommnds. Clik Defult, nd then OK. Or... Restore the Generl lyout. Clik Configurtion > Configure for Workflow > Generl. Clik OK. (optionl) You my e sked to sve method hnges. Return disply options to defult settings. In the Configurtion menu, lik eh of the Disply Options ommnds. 12 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

13 Exerise 1 Develop n quisition method Tsk 2. Determine preursor ion msses 2 Determine preursor ion msses for ll four peks. You hve determined them orretly if you find the vlues re similr to those shown in this tle:. In the Chromtogrm Results window, mke sure tht the Rnge Selet ion in the toolr is on. Clik the left mouse utton nd drg the ursor ross the first pek to produe shded region, s in the figure elow. Right-lik the shded re, nd lik Extrt MS Spetrum from the shortut menu. The system displys n verged spetrum ross the pek in the MS Spetrum Results window. The preursor mss of the first ompound, sulfmethizole, is determined to e m/z To otin single sn, doulelik the pex of the pek. If you quired the dt file using the Agilent Jet Strem Tehnology, the retention times my e different. The sulfmix01.d dt file ws quired with different olumn so your retention times re different. Close the dt file fter finding the preursor ion msses. d e f Repet step through step for the other ompounds. The preursor ion msses should mth those in the tle in step 2. Clik File > Close Dt File. When sked if you wnt to sve the results, lik No. Some ompounds form sodium (N) nd/or potssium (K) dduts s well, orresponding to M + 23 nd M + 39 msses respetively. Seeing these msses long with the M + H n mke for n esy onfirmtion of whih ion is the pseudo-moleulr ion (M + H)+. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 13

14 Exerise 1 Develop n quisition method Tsk 3. Find optimum frgmentor voltge for mximum response Tsk 3. Find optimum frgmentor voltge for mximum response Tsk 3 shows you how to rry out the optimiztion for frgmentor voltge y reting seleted ion-monitoring experiments for eh ompound within method nd setting up multiple methods with vrying frgmentor voltges. The Frgmentor Voltge for the 6490 is set utomtilly during Autotune, nd it nnot e set in the Dt Aquisition progrm. If your instrument is 6490, skip to Tsk 4. Determine produt ion msses. You n do the Qulittive Anlysis prt of this tsk y using the dt files tht were shipped with the softwre. 1 Set up six methods for six different frgmentor voltges. Chnge to SIM experiment. Use 60, 80, 100, 140, 180 nd 220 volts s the frgmentor voltges for the six methods. Sve the methods s iiims2simxxx.m, where iii re your initils nd xxx is the voltge. In the Sn Type dropdown list, lik MS2 SIM. 14 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

15 Exerise 1 Develop n quisition method Tsk 3. Find optimum frgmentor voltge for mximum response d e f In the Aquisition t, enter the Compound Nme nd Mss (preursor ion mss) for sulfdimethoxine. Right-lik nywhere in the Sn segments setion, nd lik Add Row. Type the Compound Nme nd the Mss for sulfhloropyridzine. Repet steps nd d for sulfmethzine nd sulfmethizole. Sve the method s iiims2sim140.m, where iii re your initils. g Chnge the frgmentor voltge to 60, nd sve the method s iiims2sim060, where iii re your initils. h Repet step g for voltges 80, 100, 180 nd 220, sving the methods s iiims2sim080, iiims2sim100, iiims2sim180 nd iiims2sim220, where iii re your initils. With the MS2SIM Sn Type set, different set of olumns ppers in the Aquisition window. The Instrument Control nd Dt Aquisition progrm retes SIM experiment for eh ompound mss, strting with defult frgmentor voltge of 140. See the exmple elow. The Frgmentor olumn is gryed out if the instrument type is n Agilent Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 15

16 Exerise 1 Develop n quisition method Tsk 3. Find optimum frgmentor voltge for mximum response 2 Set up nd run the worklist (optionl). Set up six smples with Smple Nme SulfDrugMix to injet 1ul from vils 1-6 or the ones you hoose. Speify the dt files s iiisulfsimxxx.d, where iii re your initils nd xxx is the voltge. d e f g Clik the Worklist ion if neessry to mke sure the worklist is visile. Clik Worklist > New to strt new worklist. You do not need to sve the lst worklist. To set up the run, right-lik the upper left orner of the worklist, nd lik Worklist Run Prmeters. Type the pths for the method nd dt files. Type the informtion for the 60 voltge run. Clik Worklist > Add Smple. Another smple is dded to the Worklist. Add five smples to the worklist for voltges Mrk the hekox to the left of the Smple Nme for eh of the six smples. This step is optionl euse you n use dt files shipped with the system to perform mny of the tsks in this exerise. h Strt the worklist. Clik Worklist > Run. Clik the ion in the min toolr. Clik the ion in the worklist toolr. Note tht the progrm only runs those smples tht re mrked with hekmrk. You n lso run the worklist in loked mode y liking the utton in the min toolr. 16 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

17 Exerise 1 Develop n quisition method Tsk 3. Find optimum frgmentor voltge for mximum response 3 Set up qulittive method to view the EIC dt utomtilly. Open the dt file Sulf_SIM60.d or your own iiisulf_sim60.d, where iii re your initils. In the Method Editor, dd in the EICs orresponding to the preursor ion msses of 271, 279, 285, nd 311. Sve the method s iiiexerise1, where iii re your initils. Clik File > Open Dt File. The system displys the Open Dt File dilog ox Selet either Sulf_SIM60.d or iiisulf_sim60.d, nd lik Open. Clik Method > Method Editor or View > Method Editor. The system displys the Method Editor window. The Qulittive Anlysis progrm should e open. If not, see Doule-lik the Qulittive Anlysis ion. on pge 11. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 17

18 Exerise 1 Develop n quisition method Tsk 3. Find optimum frgmentor voltge for mximum response d e f g If neessry, lik Define Chromtogrms in the Chromtogrm setion of the Method Explorer. To delete the BPC hromtogrm, lik Delete. Selet EIC for the Chromtogrm Definition Type, In the MS Chromtogrm t, mke sure MS Level is set to All nd Sns is set to All sn types. Cler the Do yle sum hek ox. Type 271 s the m/z vlue. h i j Clik Add. k l Repet steps i nd j for the other preursor ions, 279, 285 nd 311. Clik Method > Sve As. The system opens the Sve As dilog ox m Sve the method s iiiexerise 1.m. n Clik Sve. The defult Method Editor list seletion fter instlltion is Integrte (MS). You n lso selet Define Chromtogrms from the Method Items list in the Method Editor window. 18 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

19 Exerise 1 Develop n quisition method Tsk 3. Find optimum frgmentor voltge for mximum response 4 Extrt the hromtogrm for the dt file nd view the results. Mke sure you n see ll five hromtogrms, the TIC nd four EICs. Clik the Run utton on the Method Editor toolr. You n lso lik the Chromtogrms > Extrt Defined Chromtogrms ommnd to extrt the defined hromtogrms. To see the TIC nd four EICs, lik the rrow next to the Mximum Numer of List Pnes ion in the Chromtogrm Results toolr, s shown in the exmple elow. Selet 5 to view five hromtogrms simultneously. The system displys hromtogrm results s shown elow. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 19

20 Exerise 1 Develop n quisition method Tsk 3. Find optimum frgmentor voltge for mximum response 5 Extrt the remining ion hromtogrms utomtilly. Extrt Defined Chromtogrms should e the defult tion for Assign File Open Ations. Open the remining dt files, Sulf_SIM80.d through Sulf_SIM220.d. Close the Method Explorer. Selet File Open Ations from the Generl setion in the Method Explorer. Mke sure tht Ations to e run list only ontins Extrt Defined Chromtogrms. The Qulittive Anlysis Method Editor lets you define tions to e performed utomtilly upon opening dt file(s). d e Clik File > Open Dt File. The system displys the Open Dt File dilog ox. Selet the dt files to e opened, Sulf_SIM80.d through Sulf_SIM220.d. Mrk the Run File Open tions from seleted method hek ox. (lower left orner) 20 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

21 Exerise 1 Develop n quisition method Tsk 3. Find optimum frgmentor voltge for mximum response Mrk this hek ox. f g Clik Open. The Qulittive Anlysis progrm displys ll the EICs for ll the dt files seleted. To lose the Method Explorer nd Method Editor, lik the X in the upper right orner of eh window. You n lso lose the Method Explorer nd Method Editor windows y liking the View > Method Explorer ommnd nd the View > Method Editor ommnd. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 21

22 Exerise 1 Develop n quisition method Tsk 3. Find optimum frgmentor voltge for mximum response 22 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

23 Exerise 1 Develop n quisition method Tsk 3. Find optimum frgmentor voltge for mximum response 6 Selet the frgmentor voltge tht produes the mximum response for eh of the preursor ions. Close the dt files fter you determine the optimum voltge. In the Dt Nvigtor window, highlight the EICs for m/z. Clik the Show only the highlighted items ion,. Only the 271 m/z hek oxes re now mrked. Look t the reltive intensities of eh pek to determine whih frgmentor voltge setting will e est to use for the 271 preursor. You press the Ctrl key to e le to selet multiple ojets from the Dt Nvigtor window. You press the Shift key to e le to selet group of ojets. A frgmentor voltge of 100 should e suffiient for eh preursor ion. You n now determine the produt ions tht re ville for the multiple-retion monitoring experiments to mximize sensitivity for the nlysis. You n overly the hromtogrms y liking the Overlid mode ion in the Chromtogrm Results toolr. d e f Repet step through step for the other three se peks or preursor ions. Clik File > Close Dt File. Clik Close when the Close Dt File dilog ox ppers. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 23 Clik the different EICs in the Dt Nvigtor window to hnge whih hromtogrm is leled in the Chromtogrm Results window. When the olor of the lel of the hromtogrm mthes the olor of the hromtogrm tht hs the highest intensity, you use the frgmentor voltge tht ws used for tht file.

24 Exerise 1 Develop n quisition method Tsk 4. Determine produt ion msses Tsk 4. Determine produt ion msses In this prt of the method development, we will use three ollision energies to determine the est frgment ions to use for the eventul Multiple Retion Monitoring (MRMs) quisition. 1 Set up three produt ion quisition methods nd quire dt. Use the MS prmeters in the exmple elow, ut hnge the Frgmentor voltge to the optimum voltge you determined in the previous tsk. Sve methods s iiisulfmix PI_xx.m, where iii re your initils nd xx is the ollision energy. d e Clik the QQQ t in the Method Editor pne. Selet Produt Ion in the Sn Type omo ox to sn eh preursor ion for ll its produt ions. Enter ll MS prmeters s listed in the exmple elow, mking sure the Collision Energy is set to 15 nd the Frgmentor voltge is set to the optimum voltge determined in Tsk 3. Sve the method s iiisulfmix PI_15.m. Repet step nd step d for ollision energies of 30 nd 45. When you hnge the Sn Type in the Time Segments tle, the Sn segments tle is reset. If you wnt to opy the Sn segments to the new Sn segments tle, highlight ll of the lines in the Sn segments tle nd then right-lik the Sn segments tle nd lik Copy. After you selet new Sn Type, right-lik the Sn segments tle nd lik Pste from Clipord. You nnot opy nd pste the Sn segments tle etween ll Sn Types. 2 Set up nd run the worklist (optionl). Speify the dt files s iiisulfmix PI_xx.d, where iii re your initils nd xx is the ollision energy. d Clik the Worklist t. Add three smples to the worklist for ollision energies 15, 30 nd 45. Mrk the hek ox to the left of the Smple Nme for eh smple you re dding. Clik Worklist > Run. This step is optionl euse you n determine the produt ion msses from the dt files shipped with the system. Use the instrutions in Step 2 of Tsk 3 to set up the worklist. 24 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

25 Exerise 1 Develop n quisition method Tsk 4. Determine produt ion msses 3 Set up qulittive method to integrte nd extrt produt ion spetr. Use the dt files SulfmixPI_xx.d, where xx is the ollision energy, or your own dt files, iiisulfmixpi_xx.d. Open Method Explorer nd Method Editor. Use TICs set up for MS/MS, produt ion nd eh of the preursor ions 271, 279, 285, 311. Mke sure the MS/MS integrtor hs een seleted nd the mximum numer of peks hs een limited to the lrgest 100 peks. Add the ility to integrte nd extrt pek spetr to the file tions run upon dt opening. Sve the hnges to the urrent method. Clik the Open Dt File ion in the toolr. Selet SulfmixPI_15.d. Mke sure tht the Run File Open Ations from Speified Method hek ox is ler, nd lik Open. d Mke sure the Method Explorer nd the Method Editor windows re displyed; otherwise, lik the Method Explorer nd then Method Editor ions. e In the Chromtogrm setion in the Method Explorer window, selet Define Chromtogrms. f Delete ny existing hromtogrms in the Defined Chromtogrms list. g Selet TIC from the Type list in the Define hromtogrms setion. h For MS level, selet MS/MS. i Mrk the Do yle sum hek ox. j For Sns, selet Produt ion. k For Preursor ion m/z, type 271. l Clik the Add utton. m Repet steps j nd k for eh ion. The Qulittive Anlysis progrm should lredy e open nd ontin iiiexerise 1.m s the method. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 25

26 Exerise 1 Develop n quisition method Tsk 4. Determine produt ion msses n o From the Method Explorer in the Chromtogrm setion, lik Integrte (MS/MS). Selet MS/MS s the Integrtor seletion, if neessry. These dt files ontin MS/MS dt, so you need to modify the prmeters in the Integrte (MS/MS) setion. If the dt file ontined only MS dt, you would need to modify the prmeters in the Integrte (MS) setion. Figure 3 Integrte (MS/MS) > Integrtor T p Clik the Pek Filters t. Mke sure tht the Limit (y height) to the lrgest hek ox is mrked nd set to the vlue 100 s shown elow. Figure 4 Integrte (MS/MS) > Pek Filters t 26 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

27 Exerise 1 Develop n quisition method Tsk 4. Determine produt ion msses q r Clik Generl in Method Explorer, nd then lik File Open Ations. Selet Integrte nd extrt pek spetr from the Aville tions list nd lik to dd this to Ations to e run. Figure 5 Generl > File Open Ations t s To pply the hnges to the urrent method, iiiexerise1.m, lik the Sve Method ion. You n lso lik Method > Sve. 4 Run the qulittive method on the urrent dt file. In the Method Editor toolr, lik the Run utton,. When the Assign Ations to Run Opening A Dt File setion is displyed, the Ations to e run list is exeuted. The progrm first extrts the produt ion hromtogrms for eh preursor ion in the dt file. Next, it finds the lrgest pek in the totl ion hromtogrms, nd integrtes nd extrts pek spetr from eh integrted pek. See Figure 6 on pge 28. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 27

28 Exerise 1 Develop n quisition method Tsk 4. Determine produt ion msses Figure 6 Results for integrtion nd extrtion of pek spetr. 5 Run the File Open tions on the remining produt ion dt files. Use either the exmple files, Sulfmix PI_xx.d, or the dt files you quired in step 2. Clik File > Open Dt File. The system displys the Open Dt File dilog ox. Hold the Ctrl key nd do one of these: Selet the two dt files Sulfmix PI_30.d, nd Sulfmix PI_45.d. Selet the dt files you quired in step 2. Mrk the Run File Open tions from seleted method hek ox in the Open Dt File dilog ox, nd lik Open. After the dt files open, the Qul method first extrts the produt ion hromtogrms for eh preursor ion. Next, it finds the lrgest pek in the totl ion hromtogrms, nd integrtes nd extrts pek spetr from eh integrted pek. 28 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

29 Exerise 1 Develop n quisition method Tsk 4. Determine produt ion msses 6 Identify produt ions. View eh set of TICs nd spetr individully (e.g., 271 m/z first). Close the dt files. In the Dt Nvigtor, selet the TICs nd spetr for the 271 m/z preursor ion. Clik the Show only the highlighted items ion,. Clik View > MS Spetrum Pek List 1. d Exmine the spetr to see whih frgment ions re produed t whih ollision energies. e Repet steps to d until ll the produt ions re identified. The m/z produt ion is the most undnt of ny produt ion nd the highest signl is reorded t 15 V. This mens tht good hoie for the MRM for sulfmethizole would e > when the ollision energy is round 15 V. The pek my not e leled if the pek is too wide. f Clik the Close Dt File ion in the min toolr, nd lik Close when the dilog ox ontining the list of dt files pops up. The produt ions pper to e: Sulfmethizole > Sulfmethzine > Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide Sulfhloropyridzine > Sulfdimethoxine > 155.7

30 Exerise 1 Develop n quisition method Tsk 5. Find optimum ollision energy for MRM quisition Tsk 5. Find optimum ollision energy for MRM quisition In this tsk, you set up MRM quisition methods for the sulf drugs for different ollision energies. By exmining the spetr nd ompring pek intensities, you determine the optiml ollision energy settings for the ompounds. 1 Set up three MRM quisition methods. Use ll the MS prmeters in the exmple elow exept for the ollision energy vlue. Use ollision energies of 10, 15 nd 20. Sve methods s iiisulfmix MRM_xx.m, where iii re your initils nd xx is the ollision energy. d e f Clik the QQQ t. Set Sn Type to MRM. Enter ll MS prmeters shown in the exmple elow exept for the ollision energy vlue. In the ollision energy olumn, type 10 for eh ompound. Sve the method s iiisulfmix MRM_10.m. Repet step d nd step e for ollision energies of 15, 20, 25, 30 nd 35 sving the methods s iiisulfmix MRM_xx.m, where iii re your initils nd xx is the ollision energy. Beuse the lrgest peks were produed with ollision energy of 15 in the previous exerise, you will look t only those ollision energies to either side of Set up nd run the worklist (optionl). Speify the dt files s iiisulfmix MRM_xx.d, where iii re your initils nd xx is the ollision energy. d Clik the Worklist t to mke the worklist visile. Add six smples to the worklist for ollision energies 10, 15, 20, 25, 30, 35. Mrk the hekox to the left of the Smple Nme for eh of the three smples. Clik Worklist > Run. This step is optionl euse you n use the six exmple dt files in the next step. 30 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

31 Exerise 1 Develop n quisition method Tsk 5. Find optimum ollision energy for MRM quisition 3 Compre the ompound trnsition intensities t different ollision energies. Open the MRM dt files: SulfmixMRM_10.d SulfmixMRM_15.d SulfmixMRM_20.d SulfmixMRM_25.d SulfmixMRM_30.d SulfmixMRM_35.d Set the MRM hromtogrm extrtion prmeters s shown t right for ll trnsitions. Disle the TICs for lrity nd exmine the pek intensities. Compre the intensities of eh ompound trnsition otined t one ollision energy with the sme ompound trnsition otined t nother ollision energy. (Do this in Overlid Mode with ll the MRM hromtogrms.) Close the dt files ut don t sve results. Refer to Tle 4 on pge 32 for optiml method settings for eh ompound. d e f g Open the Qulittive Anlysis progrm. Cler the Run File Open tions... hek ox. Open the MRM dt files in the Qulittive Anlysis progrm. Right-lik the Chromtogrm Results window, nd lik Extrt Chromtogrms from the shortut menu. To selet ll dt files, lik the lst file while holding down the Shift key. Enter the prmeters s listed in the exmple elow, nd lik OK. Cler the TIC hek oxes to mke the MRM hromtogrms esier to view. Why spetrum for MRM? It s feture of the progrm to show spetr even for MRM experiments nd n e quite hndy for ompring reltive intensities of produt ions generted from the sme preursor. h Clik the Overlid Mode ion,. i Compre pek intensities for eh ompound trnsition in eh dt file in the Chromtogrm Results window. Compre the olors shown in Chromtogrm Results with the olor next to the MRM trnsition nme in the Dt Nvigtor. You n lso right-lik the Chromtogrm Results window heder nd ompre the olors of the hromtogrms to the olors of the titles in the shortut menu. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 31

32 Exerise 1 Develop n quisition method Tsk 5. Find optimum ollision energy for MRM quisition j Unless you deide to quire MRMs t lower ollision energies, you should find tht the optiml method settings re s shown in Tle 4. Clik the Close Dt File ion in the min toolr, nd lik Close when the Close Dt File dilog ox ppers. You now hve ll the informtion you need to do n MRM quisition experiment of the sulf drug mixture. Consider doing t lest one more run with those settings. Tle 4 Compounds nd Collision Energy Compounds MRM Trnsition Collision Energy (V) Sulfmethizole > Sulfmethzine > Sulfhloropyrdizine > Sulfdimethoxine > Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

33 Exerise 2 Develop Dynmi MRM quisition method from n MRM quisition dt file or n MRM method Tsk 1. Crete th file from n existing MRM dt file Exerise 2 Develop Dynmi MRM quisition method from n MRM quisition dt file or n MRM method The purpose of this exerise is to rete Dynmi MRM method from n quired MRM dt file for sulfmix_mrm dt files with the orret retention times for Dynmi MRM using the Quntittive Anlysis progrm. For this exerise, you hve three min tsks: Tsk 1. Crete th file from n existing MRM dt file on pge 33 Tsk 2. Print report in the Quntittive Anlysis progrm on pge 36 Tsk 3. Crete Dynmi MRM method using Updte dmrm on pge 37 You n esily rete Dynmi MRM method from n existing MRM method. Tsk 4. Crete Dynmi MRM method from n MRM method on pge 39 Tsk 1. Crete th file from n existing MRM dt file In this exerise, you rete th nd method from n existing MRM dt file. 1 Open the Quntittive Anlysis progrm nd rete th file with one smple file, SulfmixMRM_35.d. Copy the dt file SulfmixMRM_35.d from the instlltion disk to the \MssHunter\Dt\MRM_to_ DMRM folder. d e f g h Doule-lik the QQQ Quntittive Anlysis ion. Clik File > New Bth. Nvigte to the \MssHunter\Dt\ MRM_to_DMRM folder. Type MRM_to_DMRM in the File Nme text ox. Clik Open. Clik File > Add Smples. Selet the file SulfmixMRM_35.d. Clik OK. The file SulfmixMRM_35.d is on the instlltion disk in the \Support\Dt folder. Copy this entire folder to the \MssHunter\Dt\ MRM_to_DMRM folder. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 33

34 Exerise 2 Develop Dynmi MRM quisition method from n MRM quisition dt file or n MRM method Tsk 1. Crete th file from n existing MRM dt file 2 Crete method for tht th using MRM dt. Clik Method > New > New Method from Aquired MRM dt. Selet the SulfmixMRM_35.d dt file. Clik Open. 3 Set the Conentrtion Setup, Qulifier Setup, nd Clirtion Curve Setup. Add lirtion level 1 with onentrtion of Set the Unertinty to Reltive for ll qulifiers. Set the Curve Fit to Liner. Set the Curve Fit Origin to Inlude. Set the Curve Fit Weight to None. d e f g h i j k l Selet Conentrtion Setup in the Mnul Setup Tsks setion in the Method Tsks pne. Selet the first ompound in the tle. Right-lik the ompound row nd lik New Clirtion Level from the shortut menu. Enter 1 in the Level olumn nd 10 in the Con. olumn. Right-lik in the Level ox nd lik Copy Clirtion Levels To. Clik Selet All. Clik OK. Selet Qulifier Setup in the Mnul Setup Tsks setion in the Method Tsks pne. Verify tht the Unertinty is Reltive. Selet Clirtion Curve Setup in the Mnul Setup Tsks setion in the Method Tsks pne. Set Curve Fit to Liner for ll ompounds. Set CF Origin to Inlude for ll ompounds. Set CF Weight to None for ll ompounds. Refer to the online Help in the Quntittive Anlysis progrm for dditionl help on these tsks. 34 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

35 Exerise 2 Develop Dynmi MRM quisition method from n MRM quisition dt file or n MRM method Tsk 1. Crete th file from n existing MRM dt file 4 Verify method nd then sve the method nd pply the method to the th. d e f g Clik Method > Vlidte. Clik OK on the messge ox. Fix ny errors, if neessry. Clik Method > Sve As. Enter MRM_to_DMRM. Clik the Sve utton. Clik Method > Exit. Clik Yes to pply the method to the th. 5 Anlyze nd sve the th. Clik Anlyze > Anlyze Bth. Clik File > Sve Bth. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 35

36 Exerise 2 Develop Dynmi MRM quisition method from n MRM quisition dt file or n MRM method Tsk 2. Print report in the Quntittive Anlysis progrm Tsk 2. Print report in the Quntittive Anlysis progrm In this tsk, you print report using ny templte. You n updte Dynmi MRM method using either dt file or quntittion report folder, so this tsk retes the quntittion report folder. 1 Print report using the templte MRM_to_DMRM.xltx. d e Clik File > Sve. Clik Report > Generte. The system displys the Report dilog ox. Selet the Templte file. Selet the Report folder. This folder nme will e used in the next tsk. Clik OK. Copy the MRM_to_DMRM.xltx templte from the \Support\Dt folder on the instlltion disk. For this report, you do not need to print the report. You need to lik Advned to selet different printer. If you don t wnt to print this report, lik Advned insted. 2 Chek the sttus of the report using the Queue Viewer progrm. Clik Report > Queue Viewer. Wit for the report to finish printing. Close the Tsk Queue Viewer progrm. 36 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

37 Exerise 2 Develop Dynmi MRM quisition method from n MRM quisition dt file or n MRM method Tsk 3. Crete Dynmi MRM method using Updte dmrm Tsk 3. Crete Dynmi MRM method using Updte dmrm You n rete Dynmi MRM method from n MRM dt file or Quntittive Anlysis method. You first set the Sn Type to Dynmi MRM, nd then you use the Updte MRM Method dilog ox. 1 Open the method iiisulfmix MRM_10.m nd sve it to new nme with the formt iiisulfmix dmrm.m, where iii re your initils. d Clik File > Open > Method. Selet the iiisulfmix MRM_10.m method. Clik OK. Clik Method > Sve As. Type the new method nme with the formt iiisulfmix_dmrm.m. In this exmple, the th is in the \MssHunter\Dt\ MRM_to_DMRM folder. 2 Chnge the method to dynmi MRM method with the sme ompounds. You n either use dt file or the report tht ws generted in the lst tsk. d e f Clik the Aquisition t in the QQQ t in the Method Editor window. Right-lik the Sn segments tle nd lik Updte MRM Method. The Updte MRM Editor dilog ox opens. Selet the folder ontining the report.results.xml file or the dt file iiisulfmix MRM_10.d. Selet True for Updte Retention Time?. Selet True for Add new Compound. Clik OK. The Updte MRM Method tool utomtilly sets the Sn type to Dynmi MRM. You n selet either dt file tht ws quired with Sn Type of MRM or Qunt Report folder s the input to this dilog ox. The Sn segments re reted from one of these two input soures. You n updte the ompounds in the Sn segments tle y using QQQ dt file or Quntittive nlysis report folder. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 37

38 Exerise 2 Develop Dynmi MRM quisition method from n MRM quisition dt file or n MRM method Tsk 3. Crete Dynmi MRM method using Updte dmrm The ompounds from the dt file or quntittion report re utomtilly dded to the Sn segments tle. g h i j Selet the originl ompound in the Sn segments tle. Right-lik the row nd lik Delete Row. Verify tht eh row hs Compound Nme. A lnk Compound Nme is not llowed. Clik Method > Sve. 38 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

39 Exerise 2 Develop Dynmi MRM quisition method from n MRM quisition dt file or n MRM method Tsk 4. Crete Dynmi MRM method from n MRM method Tsk 4. Crete Dynmi MRM method from n MRM method You n rete Dynmi MRM method diretly from n MRM method y using the Pste from Clipord ommnd from the shortut menu. 1 Open the method iiisulfmix MRM_10.m nd sve it to new nme with the formt iiisulfmix dmrm_esy.m, where iii re your initils. d e f Clik File > Open > Method. Selet the iiisulfmix MRM_10.m method. Clik OK. Clik Method > Sve As. Type the new method nme with the formt iiisulfmix_dmrm2.m. Clik the Sve utton. 2 Copy ll ompounds from the Sn segments tle in the MRM method. Clik the Aquisition t in the QQQ t in the Method Editor. Selet ll of the rows in the Sn segments tle. Right-lik the Sn segments tle nd lik Copy. To selet ll of the rows in the Sn segments tle, you selet the first row in the tle, Then, you sroll to the lst row in the Sn segments tle. Press the Shift key nd selet the lst row in the tle. 3 Chnge the Sn Type to Dynmi MRM nd pste the rows into the new Sn segments tle. d e Selet Dynmi MRM s the Sn Type. Right-lik the Sn segments tle nd lik Pste from Clipord. Selet the originl ompound in the Sn segments tle. Right-lik nd lik Delete Row. Clik Method > Sve. To omine multiple Time Segments into one Dynmi MRM Time Segment, you pste the Sn segments into Exel nd rete one long list. When ll of the sn segments hve een psted into Exel, then opy ll of the Sn segments in Exel. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 39

40 Exerise 3 Crete Triggered Dynmi MRM quisition method Tsk 1. Crete Triggered Dynmi MRM method from Dynmi MRM method mnully Exerise 3 Crete Triggered Dynmi MRM quisition method For this exerise you nlyze mixture of four sulfonmide ompounds. Tsk 1. Crete Triggered Dynmi MRM method from Dynmi MRM method mnully You n rete Triggered Dynmi MRM method diretly from Dynmi MRM method. In Triggered Dynmi MRM method, you speify some of the trnsitions to e primry trnsitions. These trnsitions re quired for the entire retention time window. Some of these primry trnsitions re lso mrked s triggers. As the dt is quired, the softwre heks whether or not the undnes of the trigger trnsitions re higher thn the threshold. If the undnes re higher thn the thresholds nd other dditionl onditions re met, then the seondry trnsitions re quired. These other onditions re desried in the Conepts guide. 1 Open the method iiisulfmix MRM_10.m nd sve it to new nme with the formt iiisulfmix dmrm_esy.m, where iii re your initils. d e f Clik File > Open > Method. Selet the iiisulfmix_dmrm2.m method. Clik OK. Clik Method > Sve As. Type the new method nme with the formt iiisulfmix_triggereddmrm.m. Clik the Sve utton. A Triggered Dynmi MRM method is type of Dynmi MRM method. The Sn Type for oth methods is Dynmi MRM. The Dynmi MRM method is the templte method for the optimiztion. 40 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

41 Exerise 3 Crete Triggered Dynmi MRM quisition method Tsk 1. Crete Triggered Dynmi MRM method from Dynmi MRM method mnully 2 Chnge the method to triggered dynmi MRM method. d Clik the Aquisition t in the QQQ t in the Method Editor. Mrk the Triggered hek ox in the Triggered MRM setion. This setion is only ville if the Sn Type is Dynmi MRM. Selet whether to utomtilly mrk the highest produt ion s the Primry. Enter the vlue for Repets. Severl olumns re dded to the Sn segments tle. These olumns only pply to triggered dynmi MRM method. The vlue Repets is the numer of times to quire eh of the seondry trnsitions when the triggering onditions re met. 3 Selet the trnsitions tht re the Primry trnsitions. For eh trnsition, mrk the Primry hek ox if it is Primry trnsition. Verify tht you hve mrked t lest one trnsition s the Primry trnsition for eh Compound Nme. You n selet multiple trnsitions from eh ompound to e Primry trnsitions. If trnsition hs the sme Compound Nme, then it is prt of the sme ompound. You must mrk t lest one trnsition s Primry trnsition for eh ompound. 4 Selet the trnsitions tht re the Trigger trnsitions nd set the trigger onditions. d e f For eh ompound, mrk the Trigger hek ox if it is Trigger trnsition. (optionl) Mrk seond Trigger trnsition. (optionl) Enter the Threshold vlue for eh Trigger trnsition. (optionl) Enter the Trigger Entrne for eh Trigger trnsition. (optionl) Enter the Trigger Dely for eh Trigger trnsition. (optionl) Enter the Trigger Window for eh Trigger trnsition. For eh ompound, you n hve two Trigger trnsitions. If the Trigger trnsition hs n undne over the Threshold, then tht triggering ondition is met. By defult, the Trigger Entrne, the Trigger Dely nd the Trigger Window re set to 0. If these vlues re 0, then these triggering onditions re not enled. See the Conepts Guide for more informtion on these trigger onditions. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 41

42 Exerise 3 Crete Triggered Dynmi MRM quisition method Tsk 2. Add/Modify ompounds in n existing dtse Tsk 2. Add/Modify ompounds in n existing dtse You n lso mnully dd ompounds to dtse nd modify the ompounds in the dtse. In the next tsk, you rete Triggered Dynmi MRM method from the ompounds in the dtse. 1 Review the iiisulfmix_dmrm2.m, where iii re your initils. d Clik File > Open > Method. Selet the iiisulfmix_dmrm2.m method. Clik OK. Review the prmeters. A Triggered Dynmi MRM method is type of Dynmi MRM method. The Sn Type for oth methods is Dynmi MRM. 2 Strt the MssHunter Optimizer softwre. Doule-lik the Optimizer ion.. If you re optimizing peptides, use the Optimizer for Peptides progrm. 42 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

43 Exerise 3 Crete Triggered Dynmi MRM quisition method Tsk 2. Add/Modify ompounds in n existing dtse 3 Set prmeters on the Optimizer Setup t. Clik the Optimizer Setup t. Clik the Injetion (with or without olumn) utton. Set the CE rnge from 4 to 48. d Set the Cell Aelertor Voltge to 7. e Right-lik the tle nd lik Add Method. f Selet the iiisulfmix_dmrm2.m method. To rete low mss produt ions from preursor ion ner 300 m/z, you need firly high ollision energies. 4 Set prmeters on the Preursor Ion Seletion t. Clik the Preursor Ion Seletion t. Verify tht +H is mrked for the Positive ions (with priorities) list. 5 Set prmeters on the Produt Ion Seletion t. Clik the Produt Ion Seletion t. Clik the Mss (m/z) utton under Low mss ut-off. Enter 60 for the low mss ut-off. On the Produt Ion Seletion, you n utomtilly dd up to 4 produt ions per ompound (for instne, 2 primries nd 2 seondries). You wnt 8 to 10 peks in the omposite spetrum to prove tht this is inditive of the ompound, so you need to dd t lest some of the produt ions mnully. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 43

44 Exerise 3 Crete Triggered Dynmi MRM quisition method Tsk 2. Add/Modify ompounds in n existing dtse 6 Set prmeters on the Compound Setup t nd dd dditionl trnsitions. For Preursor ion 311, dd the following produt ions: 244.8, 229.7, 217.7, 172.9, 107.9, 91.9, 79.9, 64.8 For Preursor 285, dd the following produt ions: 129.9, 107.9, 91.9, 79.8, 64.8 For Preursor 279, dd the following produt ions: 212.8, 155.9, 123.9, 107.9, 91.9, 79.8, 64.9 For Preursor 271, dd the following produt ions: 177.8, 115.9, 107.9, 92, 80, 64.9 Clik the Compound Setup t. Clik the Import/Export > Import from Aquisition Methods ommnd. Selet the iiisulfmix_dmrm2.m method nd lik Open. d (optionl) Right-lik the t nd lik Expnd/Collpse All Rows. e Selet one of the Produt rows for one of the ompounds. In this exmple, selet the Produt row for Preursor 311. f Right-lik the Produt row nd lik Add Produt Ion. In this exmple, you dd 8 produt ion rows. g Enter the Produt in eh of the produt ion rows tht were dded. See To determine produt ions in the Qulittive Anlysis progrm: on pge 45. h Add produt ions for ompounds 1, 2 nd 3. For eh ompound, we re going to dd dditionl trnsitions. In the Qulittive Anlysis progrm, you exmine Produt Ion dt files whih you quired previously to determine dditionl trnsitions to dd. See Tsk 4. Determine produt ion msses on pge 24. You n use the rrow keys to move etween rows in the Produt tle. This produt ion sn hs preursor mss of 311. You exmine the MS spetrum to determine the produt ions to dd to the Produt ion setion of the Compound Setup tle. The produt ions tht re mnully dded s dditionl Produt ions in the MssHunter Optimizer softwre re shown in the MS Spetrum Results window. The green oxes were dded in this guide to show whih produt ions were used. 44 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

45 Exerise 3 Crete Triggered Dynmi MRM quisition method Tsk 2. Add/Modify ompounds in n existing dtse 3 Set prmeters on the Optimizer Setup t. Clik the Optimizer Setup t. Clik the Injetion (with or without olumn) utton. Set the CE rnge from 4 to 48. d Set the Cell Aelertor Voltge to 7. e Right-lik the tle nd lik Add Method. f Selet the iiisulfmix_dmrm2.m method. To rete low mss produt ions from preursor ion ner 300 m/z, you need firly high ollision energies. 4 Set prmeters on the Preursor Ion Seletion t. Clik the Preursor Ion Seletion t. Verify tht +H is mrked for the Positive ions (with priorities) list. 5 Set prmeters on the Produt Ion Seletion t. Clik the Produt Ion Seletion t. Clik the Mss (m/z) utton under Low mss ut-off. Enter 60 for the low mss ut-off. On the Produt Ion Seletion, you n utomtilly dd up to 4 produt ions per ompound (for instne, 2 primries nd 2 seondries). You wnt 8 to 10 peks in the omposite spetrum to prove tht this is inditive of the ompound, so you need to dd t lest some of the produt ions mnully. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 45

46 Exerise 3 Crete Triggered Dynmi MRM quisition method Tsk 2. Add/Modify ompounds in n existing dtse 6 Set prmeters on the Compound Setup t nd dd dditionl trnsitions. For Preursor ion 311, dd the following produt ions: 244.8, 229.7, 217.7, 172.9, 107.9, 91.9, 79.9, 64.8 For Preursor 285, dd the following produt ions: 129.9, 107.9, 91.9, 79.8, 64.8 For Preursor 279, dd the following produt ions: 212.8, 155.9, 123.9, 107.9, 91.9, 79.8, 64.9 For Preursor 271, dd the following produt ions: 177.8, 115.9, 107.9, 92, 80, 64.9 Clik the Compound Setup t. Clik the Import/Export > Import from Aquisition Methods ommnd. Selet the iiisulfmix_dmrm2.m method nd lik Open. d (optionl) Right-lik the t nd lik Expnd/Collpse All Rows. e Selet one of the Produt rows for one of the ompounds. In this exmple, selet the Produt row for Preursor 311. f Right-lik the Produt row nd lik Add Produt Ion. In this exmple, you dd 8 produt ion rows. g Enter the Produt in eh of the produt ion rows tht were dded. See To determine produt ions in the Qulittive Anlysis progrm: on pge 45. h Add produt ions for ompounds 1, 2 nd 3. For eh ompound, we re going to dd dditionl trnsitions. In the Qulittive Anlysis progrm, you exmine Produt Ion dt files whih you quired previously to determine dditionl trnsitions to dd. See Tsk 4. Determine produt ion msses on pge 24. You n use the rrow keys to move etween rows in the Produt tle. This produt ion sn hs preursor mss of 311. You exmine the MS spetrum to determine the produt ions to dd to the Produt ion setion of the Compound Setup tle. The produt ions tht re mnully dded s dditionl Produt ions in the MssHunter Optimizer softwre re shown in the MS Spetrum Results window. The green oxes were dded in this guide to show whih produt ions were used. 46 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

47 Exerise 3 Crete Triggered Dynmi MRM quisition method Tsk 2. Add/Modify ompounds in n existing dtse To determine produt ions in the Qulittive Anlysis progrm: d e f Open the SulfmixPI_15.d from Tsk 4. Determine produt ion msses on pge 24. Clik Find > Find Compounds y Trgeted MS/MS. Close the Compound List window. Selet ompound in the Dt Nvigtor window. For this exmple, lik Cpd 4. Clik the Autosle Y-xis in the MS Spetrum Results toolr. Right-lik nd drg to zoom in on the MS spetrum. If possile, rerrnge the windows on the sreen so you n see the Optimizer progrm nd the Qulittive Anlysis progrm t the sme time. This produt ion sn hs preursor mss of 311. You exmine the MS spetrum to determine the produt ions to dd to the Produt ion setion of the Compound Setup tle. 7 Set other prmeters in the Compound Setup t nd strt the optimiztion. You nnot perform multi-ompound run. You hve to mrk eh row in the tle to use. Mrk the hek ox in the left olumn t the top of the tle. The hek ox for every row in the tle is mrked. Cler the Perform multi-ompound run hek ox in the right olumn. Clik the Strt Optimiztion utton in the Optimizer toolr. You nnot perform multi-ompound run with the numer of trnsitions tht were dded. If you mrk this hek ox, then the Expeted pek width (se) is utomtilly set to lmost 80 seonds wide. If you ler this hek ox, then the Expeted pek width is lulted to e round 9 seonds whih is more pproprite. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 47

48 Exerise 3 Crete Triggered Dynmi MRM quisition method Tsk 2. Add/Modify ompounds in n existing dtse 8 Exmine the Optimizer Report. Exmine the Collision Energy for eh Produt Ion. Print or sve the report. As generl rule, s the Produt Ions get smller, the optiml Collision Energy gets lrger. However, when you lso exmine the undne, you n see tht if the Collision Energy is set to 48 for the smllest produt ion, the smllest produt ion n eome the dominnt pek. The ollision energies re further djusted lter in this tsk. 9 Sve the ompounds. Clik the File > Sve Compounds ommnd. 10 Import ompounds from dtse. Clik the Import/Export > Import from Dtse ommnd. The Dtse Browser progrm opens. You n lso import ompounds tht were distriuted s prt of dtse. 48 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

49 Exerise 3 Crete Triggered Dynmi MRM quisition method Tsk 2. Add/Modify ompounds in n existing dtse 11 In the Dtse Browser progrm, selet the trnsitions. d Mrk the Show All Reords hek ox. Clik the Selet top utton under Selet Trnsitions. Type 10 for the rnked trnsitions. Clik the Selet Trnsitions utton. All the trnsitions tht you typed in re visile. The tools to llow you to set up Primry trnsitions nd Seondry trnsitions re ville in this progrm. 12 In the Dtse Browser progrm, utomtilly selet the Primry trnsitions nd Trigger trnsition. In the Set top rnked trnsitions s primry ox, enter 2. Clik the Set Primries nd Trigger utton. The softwre utomtilly selets the two most undnt trnsitions s the Primry trnsitions. The softwre lso selets the most undnt trnsition s the Trigger. You n mnully selet seond Trigger trnsition. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 49

50 Exerise 3 Crete Triggered Dynmi MRM quisition method Tsk 2. Add/Modify ompounds in n existing dtse You exmine the Primry olumn nd the Trigger olumn to determine whih trnsitions re seleted. You n selet one or two Trigger trnsitions. You n selet multiple Primry trnsitions. 13 Review the Primry trnsitions nd Trigger trnsitions. For sulfhloropyridzine, selet 285 m/z -> 156 m/z trnsition s the Primry nd Trigger trnsition. For sulfdimethoxine, selet selet 311 m/z -> 156 m/z trnsition s the Primry nd Trigger trnsition. For sulfmethzine, selet 279 m/z -> 186 m/z trnsition s the Primry nd Trigger trnsition. For sulfmethizole, selet 271 m/z -> 156 m/z trnsition s the Primry nd Trigger trnsition. Review eh ompound. Chnge the Primry nd Trigger trnsitions to the trnsitions listed in the left olumn. Chnge the other Primry trnsitions s shown elow. The softwre seleted the most undnt trnsitions whih in this exmple often hd low m/z for the Produt Ion. A very undnt low m/z ion my e unsuitle s Primry trnsition. You n selet two Primry trnsitions s triggers for ompound. 50 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

51 Exerise 3 Crete Triggered Dynmi MRM quisition method Tsk 2. Add/Modify ompounds in n existing dtse 14 Review the Import List tle on the Import List t. d Clik the Add to Import List utton. Clik the Import List t. Review the Import List tle. Clik the Import utton. In this exmple, you re importing from the dtse to the Import List. Then, you re importing from Dtse Browser to Optimizer. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 51

52 Exerise 3 Crete Triggered Dynmi MRM quisition method Tsk 2. Add/Modify ompounds in n existing dtse 15 Review the Compound Setup tle in Optimizer. You reple ll ompounds with the ompounds from the Dtse Browser progrm. Clik the Yes to All utton. In the Compound Setup t in the Optimizer progrm, review the ompounds. The ompounds in the Optimizer progrm re overwritten y the ompounds tht you updted in the Dtse Browser progrm. 52 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

53 Exerise 3 Crete Triggered Dynmi MRM quisition method Tsk 2. Add/Modify ompounds in n existing dtse 16 Sve the new ompound prmeters to the dtse. Clik the File > Sve Compounds ommnd to sve ll of the hnges to the dtse. You nnot see these results y defult, ut the Primry nd Trigger trnsitions re updted in the projet. The Primry olumn, Trigger olumn, Trigger Entrne Dely olumn, Trigger Dely olumn, Trigger Window olumn nd Trigger MRM Threshold olumn re ville in the Compound Setup t, ut they re hidden y defult. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 53

54 Exerise 3 Crete Triggered Dynmi MRM quisition method Tsk 3. Crete Triggered Dynmi MRM method from n existing dtse Tsk 3. Crete Triggered Dynmi MRM method from n existing dtse You n rete Triggered Dynmi MRM method from dtse suh s the Pestiides or Forensis/Tox dtse. These dtses n e purhsed from Agilent. You n lso opy the informtion from n Exel spredsheet, ut tht method is not desried in this guide. 1 In the Dt Aquisition progrm, you now import the updted ompounds from the dtse. These ompounds hve optimized ollision energies nd lso Primry nd Trigger trnsitions mrked. d e f g h i j k l Swith to the Dt Aquisition progrm. Open the iiisulfmix_dmrm2.m method. In the QQQ t, lik the Aquisition t. The Sn segments tle ontins four rows whih re deleted lter. Right-lik the Sn Segments tle nd lik Import from Dtse Browser. The Dtse Browser progrm opens. Mrk the Show All Reords hek ox. Mrk ll of the trnsitions for the four sulf drug ompounds. Cler the hek oxes next to ny unwnted ompounds. Clik the Add to Import List utton. Clik the Import List t. Review the Import List tle. Clik the Import utton. Delete the originl ompounds from the Sn segments tle. Mrk the Triggered hek ox under Triggered MRM. Before you import ompounds from Dtse Browser, the Sn segments tle ontins t lest one row. After importing ompounds from the Dtse Browser, you need to remove ny originl rows. The Sn segments tle lwys hs to hve t lest one row. The triggering informtion is loded from the Dtse Browser progrm even if the Triggered hek ox is ler. See the online Help for the Dt Aquisition progrm nd the QQQ Conepts Guide for n explntion of the other triggering onditions: Trigger Entrne, Trigger Dely, nd Trigger Window. 54 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

55 Exerise 3 Crete Triggered Dynmi MRM quisition method Tsk 3. Crete Triggered Dynmi MRM method from n existing dtse 2 Sve the method to new method nme, iiisulfs_triggeropt.m, where iii re your initils. Clik the Method > Sve Method ommnd. Type iiisulfs_triggeropt.m. Clik the Sve utton. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 55

56 Exerise 3 Crete Triggered Dynmi MRM quisition method Tsk 3. Crete Triggered Dynmi MRM method from n existing dtse 3 Review the method in the Dynmi MRM Viewer dilog ox. Right-lik the Sn segments tle nd lik Edit DMRM Method. The Dynmi MRM Viewer dilog ox is opened. Type 200 for the Cyle time. This vlue is shown in the Aquisition t. Clik etween the Primries only utton nd the All trnsitions utton if the Dynmi MRM Sttistis informtion is not updting. Then, lik the All trnsitions utton. The ompounds in the Optimizer progrm were overwritten y the ompounds tht you updted in the Dtse Browser progrm. You n modify the Cyle time nd see how the Minimum Dwell Time is hnged. If the Minimum Dwell Time is less thn 5 ms, nd espeilly if it is less thn 2 ms, then signl-to-noise is poor. A Dwell Time of 8 ms per trnsition is fine. 56 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

57 Exerise 3 Crete Triggered Dynmi MRM quisition method Tsk 3. Crete Triggered Dynmi MRM method from n existing dtse 4 Review the Trigger Thresholds to verify tht they re pproprite. d e f Do n injetion to mke sure tht the Trigger Thresholds re set properly. Right-lik the Sn segments tle nd lik Updte DMRM Method. In the MRM Updte Options dilog ox, selet True for Updte Threshold?. Enter the vlue for the Perent of Height for the Trigger Threshold. Selet the dt file tht you just quired. Clik OK. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 57

58 Exerise 4 Optimize Aquisition prmeters Tsk 1. Use the Optimizer Softwre to optimize quisition prmeters Exerise 4 Optimize Aquisition prmeters For this exerise you optimize mixture of four sulfonmide ompounds. Tsk 1. Use the Optimizer Softwre to optimize quisition prmeters The Optimizer Softwre helps you optimize quisition prmeters. Speifilly, it utomtes the seletion of the est preursor ions, the optimiztion of the frgmentor voltge for eh preursor ion, seletion of the est produt ions, nd optimiztion of ollision energy vlues for eh trnsition for list of ompounds you speify. To do this tsk, you first need to rete the method iiisulfmix MRM_10.m in Tsk 5. Find optimum ollision energy for MRM quisition on pge 30. You do not need to quire the dt file. The Frgmentor Voltge for the 6490 is set utomtilly during Autotune. The Frgmentor voltge for 6490 is not optimized. The Frgmentor prmeters nd results will not e displyed for 6490 instrument. 1 Strt the MssHunter Optimizer softwre. Doule-lik the Optimizer ion.. If you re optimizing peptides, use the Optimizer for Peptides progrm. 58 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

59 Exerise 4 Optimize Aquisition prmeters Tsk 1. Use the Optimizer Softwre to optimize quisition prmeters Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 59

60 Exerise 4 Optimize Aquisition prmeters Tsk 1. Use the Optimizer Softwre to optimize quisition prmeters 2 Set the optimiztion prmeters. Clik the Optimizer Setup t. Set the Smple introdution method to Injetion. Set the Frgmentor rmp prmeters s follows: Set the rnge for rmping the Frgmentor vlues from 90 to 135. Cler the Frgmentor Fine hek ox. d Set the rnge for rmping the Collision Energy from 0 to 40 V. e Selet Pth for dt files to store the optimiztion run dt. f Right-lik the tle on the right nd selet Add Method from the shortut menu. g Clik the utton on the right side of the Aq Method ell to open the Open Method dilog ox. h Selet the method reted in the previous exerise iiisulfmix MRM_10.m nd lik OK. The Polrity nd Ion Soure will e filled in from the vlues set in the seleted method. i Chek to mke sure tht the Ion Soure from the method mthes the physil onfigurtion of your instrument. j Repet step f to step i to selet dditionl methods. Fine optimiztion refines the orse rmping vlues nd provides etter optimiztion ut tkes longer to run. The dt n e displyed lter with Agilent MssHunter Qulittive Anlysis softwre. The Frgmentor Voltge is not optimized for n Agilent 6490 Triple Qudrupole. It is set utomtilly when you Autotune. The Frgmentor prmeters nd results for 6490 re not shown in the Optimizer progrm. 60 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

61 Exerise 4 Optimize Aquisition prmeters Tsk 1. Use the Optimizer Softwre to optimize quisition prmeters 3 Selet the preursor ions Clik the Preursor Ion Seletion t. Selet the Positive ions +H ddut. Selet the Chrge stte of 1. d Set the serh priority of the preursor ions. e (optionl) To exlude ertin msses from onsidertion, lik Exlude msses t the ottom of the sreen. Enter the m/z Vlues to exlude seprted y omms nd/or enter Minimum undne vlue in ounts. Mrk the Use most undnt preursor ion hek ox to use the most undnt preursor ion. Cler the Use most undnt preursor ion hek ox nd use the Up nd Down rrow uttons to set the serh order (ions t the top of the list re given more priority). You n lso enter Neutrl Losses to exlude (for exmple H 2 O). Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 61

62 Exerise 4 Optimize Aquisition prmeters Tsk 1. Use the Optimizer Softwre to optimize quisition prmeters 4 Selet the produt ions Clik the Produt Ion Seletion t. Enter Low mss ut-off vlue. Selet Mss (m/z) of 60 m/z. To exlude ertin msses from onsidertion, lik Exlude msses option t the ottom of the sreen. Enter the m/z Vlues to exlude seprted y omms nd/or enter Minimum undne vlue in ounts. d If desired, you n lso enter Neutrl Losses to exlude, for exmple H 2 0. Enter formul in the ox nd lik the utton to dd it to the list. 62 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

63 Exerise 4 Optimize Aquisition prmeters Tsk 1. Use the Optimizer Softwre to optimize quisition prmeters 5 Set up ompound list. The formul for the four Sulf Drugs re: Sulfmethizole C 9 H 10 O 2 N 4 S 2 Sulfmethzine C 12 H 14 O 2 N 4 S Sulfhloropyridzine C 10 H 9 O 2 N 4 SCl Sulfdimethoxine C 12 H 14 O 4 N 4 S d e f g h i j k Clik the Compound Setup t. Cler the Show results summry hek ox ove the tle while you set up the ompound list. Right-lik the tle nd selet Add Compound from the shortut menu to dd row to the end of the tle. Enter Sulfmethizole s the Compound Nme. Enter Sulf drugs s the group nme in the Groups olumn. Enter C9H10O2N4S2 s the Formul of the ompound. The mss is lulted. Enter the Smple Position for the new ompound. (optionl) Enter n Optimiztion dwell time vlue to set longer or shorter yle times. Repet the steps ove to dd the other three sulf drugs to the tle. Mrk the Selet olumns for the ompounds (rows) to use for optimiztion. Sve the ompound list to the dtse or to the urrent projet. Compounds re glol to ll projets. Compound informtion suh s nme, group, formul, nd mss in one projet will e refleted in the entire dtse. If no methods or ions re speified here, then optimiztion for the ompound uses the methods from the Optimizer Setup t nd informtion from the Preursor Ion Seletion nd Produt Ion Seletion ts to generte the ions. You n lso enter the monoisotopi mss in the Mss olumn insted of the Formul. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 63

64 Exerise 4 Optimize Aquisition prmeters Tsk 1. Use the Optimizer Softwre to optimize quisition prmeters The Frgmentor prmeters nd results re not displyed for n Agilent 6490 Triple Qudrupole. The Frgmentor voltge for 6490 is set utomtilly during Autotune. 6 Strt the optimiztion proess. Clik the Strt Optimiztion utton ( ) on the toolr or Clik the Ion Brekdown Profile utton ( ) on the toolr. 7 Review results. Clik the Compound Setup t. Mrk the Show results summry hek ox ove the tle. Review the following vlues for eh trnsition ion in the Compound Tle: Frgmentor Collision Energy d Review the printed optimiztion report. (optionl) Use the Agilent MssHunter Worksttion Qulittive Anlysis progrm to look t the dt. See the online Help for the Optimizer progrm or the Optimizer Quik Strt Guide to lern how to import optimiztion results to quisition for MRM time segments. 64 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

65 Exerise 4 Optimize Aquisition prmeters Tsk 2. Use the Soure nd ifunnel Optimizer progrm to optimize quisition prmeters Tsk 2. Use the Soure nd ifunnel Optimizer progrm to optimize quisition prmeters The Soure nd ifunnel Optimizer Softwre helps you optimize quisition prmeters for the soure nd ifunnel. To do this tsk, you first need to rete the method iiisulfmix_dmrm2.m in Tsk 4. Crete Dynmi MRM method from n MRM method on pge 39. You do not need to quire the dt file. When you use this softwre, worklist for eh of the prmeters eing optimized is dded to the Study Mnger progrm. 1 Strt the MssHunter Dt Aquisition softwre nd lod the iiisulfmix_dmrm2.m method. Sve this method to new nme. d e Strt the Dt Aquisition progrm. Mke sure tht Aquisition ppers s the seletion in the Context text ox. If Tune is the seletion, lik Aquisition from the Context dropdown menu in the Como r. Lod the iiisulfmix_dmrm2.m method. Sve this method to the nme iiisulfmix_soureopt.m. View the Method Editor window. The first step is to rete templte method. This method is used when you re optimizing the soure nd ifunnel prmeters. 2 Edit the Soure prmeters. Clik the QQQ t. Clik the Soure t on the QQQ t. Modify the prmeters to the reommended strting prmeters for soure optimiztion. These prmeters re shown in the following imge. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 65

66 Exerise 4 Optimize Aquisition prmeters Tsk 2. Use the Soure nd ifunnel Optimizer progrm to optimize quisition prmeters 3 Crete templte method. Clik the Aquisition t. Selet single ion for eh ompound for the optimiztion. Sve the method. A trnsition for eh ompound is lredy inluded in the Dynmi MRM method. 4 Strt the MssHunter Soure nd ifunnel Optimizer progrm. Doule-lik the Soure Optimizer ion ( ). When this progrm strts, it utomtilly selets the defult.m method. This method is not set up for n Agilent Jet Strem soure, so no Agilent Jet Strem prmeters re shown in the Instrument prmeters tle. 66 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

67 Exerise 4 Optimize Aquisition prmeters Tsk 2. Use the Soure nd ifunnel Optimizer progrm to optimize quisition prmeters 5 Selet the templte method, iiisulfmix_soureopt.m. Clik the Browse utton. The Browse For Folder dilog ox is opened. Selet the iiisulfmix_soureopt.m method. Clik the OK utton. If the ion soure in the method is different thn the ion soure in the Instrument prmeters list, wrning messge is opened. Clik OK. The Sheth Gs Temp, Sheth Gs Flow, nd Nozzle Voltge re ll speifi to the Agilent Jet Strem. If you do not hve n Agilent Jet Strem soure, these rows re not inluded in the tle. The High Pressure RF nd Low Pressure RF re only inluded if the QQQ model is Chnge the order of the rows in the Instrument prmeters tle to the following: Ion Funnel prmeters Sheth Gs temperture nd flow Gs temperture nd flow Neulizer Cpillry Nozzle Voltge If neessry, selet the row tht shows the High Pressure RF prmeter. Drg this row to the top of the tle. Both of the Ion Funnel prmeters re moved together. Verify tht the order of the rows in your tle is s indited. The order of the prmeters in the Instrument prmeters tle is the order tht the prmeters re optimized. You wnt to optimize the prmeters tht hve the gretest effet on the soure optimiztion first. The Ion Funnel prmeters hve the gretest effet, so you move those prmeters to the top of the list. By defult, the prmeters re in the optimized list. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 67

68 Exerise 4 Optimize Aquisition prmeters Tsk 2. Use the Soure nd ifunnel Optimizer progrm to optimize quisition prmeters The High Pressure RF nd the Low Pressure RF re lwys optimized together. If you move one of these rows in the tle, the other row is lso moved. All possile omintions of the High Pressure RF nd Low Pressure RF re tried to find the optiml vlues. For this exmple, the High Pressure RF prmeter hs 8 different prmeter settings (70, 90, 110, et.). The Low Pressure RF prmeter hs 7 different prmeter settings. So, the progrm utomtilly retes 56 (8 * 7) different methods (1 for eh prmeter omintion) just for optimizing the ion Funnel prmeters. 7 Review the vlues for eh prmeter in the Instrument prmeters tle. For eh row in the tle, verify: PreWit (in minutes). Replite. StepWit (in minutes). StrtVlue. EndVlue. StepSize. When the study for eh prmeter is loded for the first time ut efore you run the first run, you wit the PreWit numer of minutes efore strting the run. Some prmeters (tht re eletroni) stilize lmost instntly (in milliseonds), so you do not need to wit. For flows nd tempertures, you wnt to hve PreWit efore you run the study. You lso wnt to wit for temperture prmeters in etween hnging the prmeter to different vlue, so you lso set the StepWit (in minutes). 8 Sve the Instrument prmeters. Clik File > Sve As (*.opt). Enter the nme for this set of instrument prmeters. Clik OK. 9 Modify the Instrument prmeters tle to only modify one prmeter for this tsk. This tsk only optimizes the Cpillry voltge. Mrk the hek ox next to the Cpillry. Cler the hek oxes next to ll of the other prmeters. For this exmple, you optimize the Cpillry. Usully, you optimize the prmeters in the order speified in the Instrument prmeters tle. 68 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

69 Exerise 4 Optimize Aquisition prmeters Tsk 2. Use the Soure nd ifunnel Optimizer progrm to optimize quisition prmeters 10 Set the Projet Folder nd the Projet Nme. Selet the Projet Folder. In this exmple, selet \MssHunter\Dt. Enter Projet Nme. (optionl) Mrk the Append timestmp hek ox. If you mrk the Append timestmp hek ox, then time stmp is utomtilly dded to the Projet Nme when you lik the Sumit utton. 11 Set the Worklist prmeters. Type the Smple Nme. Type the Smple Position. Type the Worklist position of dt file used for lirtion. If you mrk the Append timestmp hek ox, then time stmp is utomtilly dded to the Projet Nme when you lik Sumit. For eh prmeter tht is optimized, th file is reted for Quntittive Anlysis. One of the injetions is onsidered 100% of the strting vlue. The vlue of Worklist position of dt file used for lirtion sttes whih dt file to use. If you enter 1, then the dt file from the first row is used. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 69

70 Exerise 4 Optimize Aquisition prmeters Tsk 2. Use the Soure nd ifunnel Optimizer progrm to optimize quisition prmeters 12 Crete the methods nd sumit the study to the Study Mnger. Clik Crete Methods. Clik Sumit. When you lik Crete Methods, messge t the ottom of the min window sttes how mny Methods were reted, how mny Injetions re involved, nd the Estimted time. The Estimted time is only n pproximtion. The estimted time inludes the Stoptime for the method plus one minute per injetion. It does not onsider the Posttime speified in the method. Also, it does not inlude the PreWit nor the StepWit tht you entered in the Instrument prmeters tle. 13 Review the study (or studies) sumitted to Study Mnger. d Open the Study Mnger progrm. Selet row in the Pending Studies tle. Right-lik the row nd lik Edit Worklist From Study. Review the worklist in the Edit Worklist dilog ox. Clik Sve. A study is sumitted for eh prmeter tht you mrked in the Instrument prmeters tle. Only one study is reted for the High Pressure RF nd Low Pressure RF prmeters. The nme of the study is the Instrument prmeter tht is eing optimized. A seprte study is dded for eh prmeter tht is eing optimized. You n exmine or edit the worklist for the study. You right-lik the line in the Pending Studies tle nd lik Edit Worklist from Study. 70 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

71 Exerise 4 Optimize Aquisition prmeters Tsk 2. Use the Soure nd ifunnel Optimizer progrm to optimize quisition prmeters The sript tht is run t the end of the worklist retes the Quntittive Anlysis th file. 14 Modify the Study Mnger prmeters to run stndy sript when the study ompletes nd then strt the Study Mnger. d e f Clik the Settings t in the Rion. Mrk the Enle stndy sript exeution on idle hek ox. Clik the... utton to selet the sript to exeute. Selet SCP_InstrumentStndy nd lik the OK utton. Enter 1 for the Wit for time. Clik the Strt utton if neessry. When the Study Mnger is not running study for the time speified, then the sript you selet is exeuted. The nme of the study is the Instrument prmeter tht is eing optimized. A seprte study is dded for eh prmeter tht is eing optimized. You n exmine or edit the worklist for the study. You right-lik the line in the Pending Studies tle nd lik Edit Worklist from Study. Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 71

72 Exerise 4 Optimize Aquisition prmeters Tsk 2. Use the Soure nd ifunnel Optimizer progrm to optimize quisition prmeters 15 When the study ompletes, stop the Study Mnger queue nd exit from the Study Mnger progrm. 16 Open the dt in the Quntittive Anlysis progrm. d Clik the Stop > Immeditely ommnd. Close the Study Mnger progrm. Strt the Quntittive Anlysis progrm. Clik File > Open Bth. Nvigte to the lotion of the study. Selet the Bth file nmed Cpillry.th.in nd lik Open. You speified the Projet Folder nd the Projet Nme in the Soure nd ifunnel Optimizer progrm efore you sumitted the study. The th file is reted utomtilly t the end of the study. The system folder ontins ll of the methods tht were used in this study. 17 Review the Bth Tle. Swith to Multiple Compound View. Add the Are olumn to the tle. For eh ompound, right-lik the Finl Con. olumn nd lik Plot this olumn. d Exmine the Are olumn nd the Finl Con. grph to determine the est pillry voltge. e Close the Quntittive Anlysis progrm. Refer to the online Help for the Quntittive Anlysis progrm to lern how to do these tsks. In this se, ll four ompounds optimize t the sme setting. Often, different ompounds hve different optiml settings, nd you hve to ompromise. 72 Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide

73 Exerise 4 Optimize Aquisition prmeters Tsk 2. Use the Soure nd ifunnel Optimizer progrm to optimize quisition prmeters 18 (optionl) Review the dt files in the Qulittive Anlysis progrm. Strt the Qulittive Anlysis progrm. Open ll of the dt files in the study. Clik Find > Find Compounds y MRM. d Selet ll of the dt files nd lik the Find utton. e Clik the Edit > Auto-Color Mode > Single Color per Dt File menu item. f Cler the hek oxes next to the TIC for eh dt file. g Exmine the results in the Chromtogrm Results window. h Close the Qulittive Anlysis progrm. By defult, the progrm selets different olors for different trnsitions. It is ler tht the onditions used for the lue hromtogrms re the est, nd the lue hromtogrms re for the dt file with the pillry voltge set to Agilent 6400 Series Triple Qud LC/MS Fmiliriztion Guide 73