_— /SCIEX) The Power of Precision Customer Familiarization Checklist for 3500, 4500, 5500, 5500+, and 6500+ Series Systems Introduction to LC-MS/MS OperationThis document is provided to customers who have purchased SCIEX equipment to use in the operation of such SCIEX equipment. This document is copyright protected and any reproduction of this document or any part of this document is strictly prohibited, except as SCIEX may authorize in writing, Software that may be described in this document is furnished under a license agreement. It is against the law to copy, modify, or distribute the software on any medium, except as specifically allowed in the license agreement. Furthermore, the license agreement may prohibit the software from being disassembled, reverse engineered, or decompiled for any purpose. 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Ltd 81k33, #04-08 Marsiling Industrial Estate Road 3 Woodlands Central Industrial Estate, Singapore 739256 Customer Familiarization Checklist for 3500, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 2183Contents 1 Familiarization. Scenarios. Objectives for the Primary Learner.. Methods. Verification of Training Requirements (10 min). System Safety (5 min). Hardware (15 min). Workflows (5 min) Analyst Software (30 min) Hands-On Lab Exercise (150 min)... Routine Maintenance (30 min) Mass Spectrometer lon Source Maintenance... Routine Cleaning of the Curtain Plate and Orifice Plate, Qualified Maintenance Person Familiarization (10 min) Documentation, Support, and Teining (10min). Training and Online Leaming. Product Information and Documentation, Technical and Warranty Support. 2 Signoff. Comments. 27 A Required Materials. Materials Supplied with the Purchase of a New System Customer-Supplied Materials... Customer-Supplied Lab Equipment. B Hands-On Lab Exercise on LC-MS/MS Operation. Preparation. Guidelines. Prepare the Mobile Phases Using Pre-Blended Solvents, 30 Prepare the Wash Solvent for the Autosampler and Syringe Cleaning. 31 Prepare the Triazine Dilutions. 32 Prepare the Infusion Dilution for Optimization (Method Developer Workflow). 33 Change the Mobile Phases. 34 Configure the System for Infusion. 34 Optimize for the Compound (Method Developer Workflow). 35 Plumb the LC System and Load the Samples. 39 Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 3183Contents Acquisition. 40 Create the Acquisition Method (Method Developer Worktiow). 40 Equilibrate the System, a2 Greate the Acquisition Batch. 44 ‘Submit the Batch. aT Review Acquired Data in Explore Mode. AB Data Processing (MultiQuant Software), 49 Build a Quantitation Method (Method Developer Worktiow). 49 Create a Results Table 51 Review the Results Table, 52 Data Processing (SCIEX OS). 56 Build a Processing Method (Method Developer Workflow). 57 Create a Results Table 61 Review the Results Table, 61 ¢ Le Methods.. ExionLC Systems. 87 Agilent 1260 and 1290 Systems, 70 Agilent 1260 and 1290 Infinity I! Systems. 73 Shimadzu Systems 75 D Additional Resources.. Hardware Software. Operating System Software, 79 Configure Mode, 80 Tune and Calibrate Mode. 81 Quantitate Mode. 81 Reporter Software. Bt Maintenance 82 Front-End Full Cleaning 82 E Alternative Solvent Preparation Metho Customer Familiarization Checklist for 3600, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 4/83 RUO-ID 8-6776-HFamiliarization 1 This Customer Familiarization is intended for use with a 3500, 4500, 5500, 5500+, or 6500+ series system running the Analyst Software. The Customer Familiarization process is intended to familiarize the primary learner with the LC-MS/MS system. The primary learner practices using the system by following the step-by-step ‘exercises from the Introduction to LC-MS/MS Operation Series for SCIEX Triple Quadrupole and QTRAP Systems eLearning, The process is intended to provide hands-on operation for the primary learner. The laboratory manager must designate a primary leamer. The primary leaner will be the focus of the familiarization, and will have the opportunity to perform the hands-on exercises. In addition, Up to two observers will be accommodated Note: Before beginning the familiarization, the primary learner must have validated the customer account by following the directions in the e-mail received from SCIEX, and then completed the assigned online training at SCIEX University™. A certificate of completion must be available Failure to complete the required Introduction to LC-MS/MS Operation Series for SCIEX Triple Quadrupole and QTRAP Systems eLeaming before the arrival of the Field Service Employee (FSE) will result in forfeit of the hands-on exercise during the on-site familiarization by the FSE. The Customer Familiarization in this checklist completes the first phase of the learning experience for the primary learner. When the familiarization is complete, the primary learner can log back on to SCIEX University™ at training.sciex.com to continue learning. This Customer Familiarization Checklist typically requires a minimum of four hours to complete. Estimated duration is indicated for each section of the checklist. Actual duration will vary depending on both the level of experience of the primary learner and the options installed. Topics covered will vary depending on the system configuration Tip! Many of the topics listed in this document are available as supplemental online eLearning modules. To access them, go to SCIEX University™. Registration is required to access this web page. Scenarios This section can be used in the following scenarios: Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 5/83+ New installation of a SCIEX Low Flow LC, SCIEX MS MicroLC, Eksigent NanoLC™ System or NanoLC™ System: The mass spectrometer Customer Familiarization is completed, except for the Hands-On Lab Exercise. The FSE completes the Customer Familiarization Checklist for the LC system instead. Refer to the Customer Familiarization Checklist for the LC system. + Eksigent NanoLC™ System, M3 MicroLC or MS MicroLC Systems used with the OptiFlow Turbo V Ion Source: The mass spectrometer Customer Familiarization is completed, except for the Hands-On Lab Exercise, The FSE completes the Customer Familiarization Checklist for the NanoLC™ Systems, M3 MicroLC System, or M5 MicroLC System instead. Refer to the Customer Familiarization Checklist for the NanoLC™ Systems, M3 MictoLC System, or MS MicroLC System + LC system not installed by SCIEX at the time of the mass spectrometer installation: The FSE is not responsible for verifying the functionality of the LC system. The following topics are not covered + Plumb the LC System and Load the Samples + Create the Acquisition Method (Method Developer Workflow) + Create the Acquisition Batch + Submit the Batch ‘Sample preparation procedures will be completed, and a set of example data will be used to perform the data analysis. Objectives for the Primary Learner + Become familiar with all safety documentation and information resources. + Prepare mobile phases and standards. + Set up the LC-MS/MS system + Perform optimization on a standard compound + Perform LC-MS/MS analysis. + Process and report data Methods The topics are addressed using one of these methods, as prescribed: + Reference: The FSE tells the customer where to find the information + Familiarize: The FSE briefly describes the concept or explains how to complete the task, and then directs the customer to reference material for more information. Customer Familiarization Checklist for 3500, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 6/83+ Perform: The FSE briefly demonstrates how to complete the task. + Practice: Customers practice the step-by-step exercises in the Introduction to LC-MS/MS Operation Series for SCIEX Triple Quadrupole and QTRAP Systems eLearning Note: The FSE will conduct this familiarization with up to three customer representatives, Only one primary learner will have the opportunity to complete the hands-on exercises, Verification of Training Requirements (10 min) The primary learner shows the course certificate for the Introduction to LC-MS/MS Operation Series for SCIEX Triple Quadrupole and QTRAP Systems eLearning, available on SCIEX University™. Ifan eLearning certificate is not shown, then the primary learner forfeits the hands-on exercises in the Customer Familiarization Checklist. The FSE will only demonstrate the exercise. Note: The certificate must contain the primary learer name and course name. Method Topic Resource Complete | NIA Perform Collection of required [+ Required Materials. = materials Practice [Access to online ‘SCIEX University™ website. = courses 4. Log on to SCIEX University™ 12. Show the completion of the Introduction to LC-MS/MS Operation Series for SCIEX Triple Quadrupole and QTRAP Systems training-portal series under Course Transcript. 3. Search within SCIEX University™ and sciex.com for information. Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 7183System Safety (5 min) This section provides background information about the risks and safety precautions required when working with the SCIEX LC-MS/MS system. Method Topic Resource Complete | N/A Reference Where to find safety information + System User Guide for the mass spectrometer. Customer Familiarization Checklist for 3500, 4500, 5500, 5500+, and 6500+ Series Systems 8/83 Introduction to LC-MS/MS OperationHardware (15 min) Ionization Techniques for SCIEX sources Method Topic Resource Complete | N/A Familiarize — | An overview ofthe mass]* SCIEX University™ = spectrometer, including:| | Customer Familiarization + Indicator LEDs + System User Guide for the (panel symbols) mass spectrometer + Diverter valve + Syringe pump + Communications + Mass spectrometer convenience switch + RESET and VENT buttons + Gas connections + Vacuum interface Familiarize [lon source overview + OptiFlow Turbo V lon = + Anoverview ofthe | Source Operator Guide standard ion source, |+ Turbo V lon Source including the probes | Operator Guide and electrodes. SCIEX University”: + For systems Introduction to the OptiFlow purchased with the | Quant Solution optional OptiFlow Turbo V lon Source, an overview of the OptiFlow Turbo V lon Source, including the probes. Reference + SCIEX University”: oO O° Introduction to LC-MS/MS Operation RUO-IDV-08- 6776-4 Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems 9183Method Topic Resource Complete | NIA How to transfer methods from other SCIEX mass spectrometer systems + Ion source parameters must be re-optimized for different ion source types. Workflows (5 min) ‘Method Topic Resource Complete | N/A Reference — |Workflows for the mass|+ System User Guide for the = spectrometer mass spectrometer + Instrument setup + Sample acquisition + Routine analysis + Method developer Customer Familiarization Checklist for 3500, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 10/83Familiarization Analyst Software (30 min) This section describes routine calibration of the mass spectrometer using the chemical standards available from SCIEX. spectrometer using manual tuning Method Topic Resource Complete | N/A Familiarize [Verify calibration with + SCIEX University™ = chemical standards by | Customer Familiarization infusion using the Instrument Optimization function Reference Optimize the mass. + System User Guide for the = mass spectrometer + SCIEX University™: Analyst’ Software — Tune and Calibrate Hands-On Lab Exercise (150 min) This section provides basic LC-MS/MS operator familiarization using a chemical standard and analytical column. For more information, refer to the Introduction to LC-MS/MS Operation Series for SCIEX Triple Quadrupole and QTRAP Systems, available on SCIEX University™. Note: Quantitation tasks can be performed with either SCIEX OS or the MultiQuant Software, depending on which the customer has purchased and installed, Introduction to LC-MS/MS Operation RUO-IDV-08-6776-H Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems 11/83Familiarization Method Topic Resource Complete | NIA Reference Safety precautions for |* SCIEX University": = the LC-MS/MS System | Introduction to LC-MS/MS Operations Procedure | Operation Series for SCIEX| Triple Quadrupole and QTRAP Systems Certificate of completion Practice How to prepare mobile |+ SCIEX University™: - phases and perform Introduction to LC-MS/MS dilutions for basic Operation Series for SCIEX| LC-MS/MS operation | Triple Quadrupole and QTRAP Systems + Hands-On Lab Exercise on LC-MS/MS Operation Practice How to plumb the LC + SCIEX University™” = system and load the Introduction to LC-MS/MS samples Operation Series for SCIEX| Triple Quadrupole and QTRAP Systems + Hands-On Lab Exercise on LC-MS/MS Operation Practice How to create an + SCIEX University™: = optimized infusion Introduction to LC-MS/MS method using Operation Series for SCIEX| compound optimization] Triple Quadrupole and QTRAP Systems + Hands-On Lab Exercise on LC-MS/MS Operation Practice How to modify the [+ SCIEX University™ = optimized MS method | Introduction to LC-MS/MS for use with an LC Operation Series for SCIEX| system Triple Quadrupole and QTRAP Systems + Hands-On Lab Exercise on LC-MS/MS Operation Customer Familiarization Checklist for 3600, 4500, 5500, 5500+, and 6500+ Series Systems 12183 RUO-IDV: Introduction to LC-MS/MS Operation 6776Method Topic Resource ‘Complete Practice [Howto create and |» SCIEX University™ = submit a batch for an | Introduction to LC-MS/MS example calibration Operation Series for SCIEX| curve Triple Quadrupole and QTRAP Systems + Hands-On Lab Exercise on LC-MS/MS Operation + System User Guide for the mass spectrometer Practice [Howto manage the |» SCIEX University™ = queue including Introduction to LO-MS/MS . Operation Series for SCIEX| Resubmit a patch Triple Quadrupole and + Delete samples QTRAP Systems + Delete abatch + Hands-On Lab Exercise on LC-MS/MS Operation + System User Guide for the mass spectrometer Practice [Howto review acquired» SCIEX University™ = data in Explore mode, in the Analyst Software + Open a data file + Review XICs + Navigate between samples Introduction to LC-MS/MS Operation Series for SCIEX Triple Quadrupole and QTRAP Systems Hands-On Lab Exercise on LC-MS/MS Operation System User Guide for the mass spectrometer Introduction to LC-MS/MS Operation RUO-IDV-08- 6776-4 Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems 13/83Method Topic Resource ‘Complete NIA. Practice [Howto review acquired|+ SCIEX University™ = data in the Explorer Introduction to LO-MS/MS workspace in Operation Series for SCIEX| SCIEX OS Triple Quadrupole and + Open a data fle QTRAP Systems Hands-On Lab Exercise on Review XICs LC-MS/MS Operation . Neve between |. software User Guide for SCIEX OS Practice | How to perform + SCIEX University™ = quantitation: Build a quantitation method in the MultiQuant software Build a processing method in the Analytics workspace of SCIEX OS Create a Results Table Review data and generate a calibration curve Generate a report Introduction to LC-MS/MS Operation Series for SCIEX Triple Quadrupole and QTRAP Systems Hands-On Lab Exercise on LC-MS/MS Operation SCIEX University™ Quantitative Data Processing Using Analyst Software ‘MultiQuant Software Reference Guide Software User Guide for SCIEX OS Customer Familiarization Checklist for 3600, 4500, 5500, 5500+, and 6500+ Series Systems 14183 RUO-IDV: Introduction to LC-MS/MS Operation 8-6776-HRoutine Maintenance (30 min) This section provides instructions for routine maintenance of the mass spectrometer system. Specific maintenance requirements for OEM components are not part of this checklist. Note: Routine maintenance is the responsibility of the customer. Maintenance services are available from SCIEX. Method — [Topic Resource Complete | N/A Reference [Essential MS Kit and www.sciex.com = Essential LC Kit. These | Search for keyword: Essential kits contain primary parts + FSE recommended for onsite stock to reduce system downtime. Review [How to find out which parts are consumable. Examples of consumable parts include: + FSE ‘sciex.com/service-consumables = + Parts and Equipment Guide + Air filters + Electrodes for the ion source probes + Turbo heaters for the ion source + Standards chemical kits + Cleaning kits + Interface heaters + Roughing pump oil Reference [Where to find masses for|* System User Guide for the = calibration ions mass spectrometer Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 15/83Mass Spectrometer Method Topic Resource Complete | NIA Familiarize [How to start up (pump |+ System User Guide for the = down) the system and | mass spectrometer how to shut down (vent) the system in an emergency Familiarize [Howto inspect the ol _|+ System User Guide for the = level for the roughing | mass spectrometer pump Familiarize — [How to restart the + Analyst Software Advanced = AnalystService User Guide + Analyst Software Help Familiarize — [How to restart the + Windows Help oO © ClearCore2 Service for |, pgp SCIEX OS Customer Familiarization Checklist for 3600, 4500, 5500, 5500+, and 6500+ Series Systems 16/83 RUO-IDV: Introduction to LC-MS/MS Operation 8-6776-Hlon Source Maintenance WARNING! Personal Injury Hazard. Always wear protective eyewear when handling the NANO column heater, A portion of the fused silica tubing protrudes from the top of the column heater and can cause injury. Method Topic Resource Complete NA Familiarize lon source safety * DuoSpray™ lon Source = information related to | Operator Guide electrical shock hazards). jenprive Turbo V lon and hot surface Source Operator Guide warnings + Turbo V lon Source Operator Guide + OptiFlow Turbo V lon Source Operator Guide Perform How to remove the ion |+ DuoSpray™ lon Source = source from the mass spectrometer Operator Guide lonDrive Turbo V lon Source Operator Guide Turbo V lon Source Operator Guide OptiFlow Turbo V lon Source Operator Guide SCIEX University™ Front End Cleaning of SCIEX Triple Quadrupole and QTRAP" Systems SCIEX University Quick Maintenance Videos for the OptiFlow Turbo V lon Source Introduction to LC-MS/MS Operation RUO-IDV-08- 6776-4 Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems 17183Topic Resource ‘Complete NIA. How to remove and install the probe (Not applicable to the OptiFlow Turbo V lon Source) | , How to adjust the position of the corona discharge needle How to replace the electrode in the probe DuoSpray™ lon Source Operator Guide JonDrive Turbo V lon Source Operator Guide Turbo V lon Source Operator Guide OptiFlow Turbo V lon Source Operator Guide SCIEX University”: onization Techniques For SCIEX sources ‘SCIEX University™ Quick Maintenance How To Videos for SCIEX Triple Quadrupole and QTRAP * Systems SCIEX University™ Quick Maintenance Videos for the OptiFlow Turbo V lon Source Customer Familiarization Checklist for 3600, 4500, 5500, 5500+, and 6500+ Series Systems 18/83 RUO-IDV: Introduction to LC-MS/MS Operation 8-6776-HMethod Topic Resource ‘Complete Perform How to install the ion’ source on the mass spectrometer DuoSpray™ lon Source Operator Guide + fonDrive Turbo V fon Source Operator Guide + Turbo V lon Source Operator Guide + OptiFlow Turbo V lon Source Operator Guide + SCIEX University™: Front End Cleaning of SCIEX Triple Quadrupole and QTRAP* Systems + SCIEX University™: Quick Maintenance Videos for the OptiFlow Turbo V lon Source Perform How to replace the sample tubing DuoSpray™ lon Source Operator Guide + lonDrive Turbo V lon Source Operator Guide + Turbo V lon Source Operator Guide + OptiFlow Turbo V lon Source Operator Guide Familiarize How to make sure that the source exhaust waste is draining properly, and how to empty the source exhaust drain bottle ‘System User Guide for the mass spectrometer Introduction to LC-MS/MS Operation RUO-IDV-08- 6776-4 Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems 19/83Familiarization Routine Cleaning of the Curtain Plate and Orifice Plate Method Topic Resource ‘Complete NIA. Perform How to remove the curtain plate + System User Guide for the mass spectrometer + SCIEX University™: Front End Cleaning of SCIEX Triple Quadrupole and QTRAP” Systems + Instrument Front-End Cleaning Procedure, available on sciex.com Familiarize How to clean the curtain plate (following the Customer Cleaning procedure) ‘System User Guide for the mass spectrometer SCIEX University™: Front End Cleaning of SCIEX Triple Quadrupole and QTRAP” Systems + Instrument Front-End Cleaning Procedure, available on sciex.com Familiarize How to clean the orifice plate (following the Customer Cleaning procedure) ‘System User Guide for the mass spectrometer ‘SCIEX University™: Front End Cleaning of SCIEX Triple Quadrupole and QTRAP” Systems + Instrument Front-End Cleaning Procedure, available on sciex.com Customer Familiarization Checklist for 3600, 4500, 5500, 5500+, and 6500+ Series Systems 20183 RUO-IDV: Introduction to LC-MS/MS Operation 6776Familiarization Qualified Maintenance Person Familiarization (10 min) Method Topic Resource Complete | N/A Familiarize (If requested by the | Qualified Maintenance Person oO ® customer) Guide Familiarization of the Qualified Maintenance Person (QMP). Customer must be made aware of the electrical and chemical risks associated with procedures in the Qualified Maintenance Person Guide. QMP Participants I, the designated QMP, have been made aware of the electrical and chemical risks associated with servicing laboratory equipment. | have been given access to the Qualified Maintenance Person Guide. Name Signature Date (yyyy-mm-dd) Name Signature Date (yyyy-mm-dd) Name Signature Date (yyyy-mm-dd) Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 24183Documentation, Support, and Training (10 min) This section provides information about available documentation resources, training resources, and online resources for on-going development, as well as for service and support. Training and Online Learning Method Topic Resource Complete | N/A Familiarize | How to register for + SCIEX University™ = classroom training Reference [Where to find the + Start > SCIEX Analyst > = Analyst Software Analyst Documentation tutorials, and what > Tutorials {topics they cover Perform Continuing with online |* SCIEX University™ = education Product Information and Documentation Method Topic Resource Complete NIA Reference |Where to find documentation for the mass spectrometer, including the System User Guide + Customer Reference DVD for the mass spectrometer Reference | Where to findion source documentation Operator Guide for the ion source, available on the ion source Customer Reference DVD Reference [Where to find documentation on Analyst Software operation + System User Guide for the mass spectrometer + Start > SCIEX Analyst > Analyst Documentation > Software Guides Customer Familiarization Checklist for 3500, 4500, 5500, 5500+, and 6500+ Series Systems 22183 Introduction to LC-MS/MS OperationMethod Topic Resource Complete | NIA Reference | Where to find + SCIEX OS Software User oO O° documentation on Guide SCIEX OS operation Start > SCIEX OS > SCIEX OS Documentation + SCIEX OS Help System Reference [Where to find the + Software installation DVD = Release Notes for the + Start > SCIEX Analyst > Analyst Software Analyst Documentation Reference | Where to find the + Software installation DVD Oo fo) Goleaee ores for Start > SCIEX OS > SCIEX OS Documentation Reference | Where to find + Peripheral Devices Guide = information cipported | * Start> SCIEX Analyst Connecting suppore > Analyst revices Documentation > Software Guides Reference | Additional basic system|+ Additional Resources in the = information Appendix Reference | Where to find + sciex.comiproductsecurity = information on general security guidance for SCIEX acquisition and processing computers. Introduction to LC-MS/MS Operation RUO-IDV-08- 6776-4 Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems 23183al and Warranty Support Method — | Topic Resource Complete | N/A Practice Registration of the mass |- SCIEX Now™ = spectrometer serial number Reference |About the StatusScope |+ StatusScope Remote Oo © Remote Monitoring Monitoring Service User Service Guide Perform — |How to use the + StatusScope Remote oO © StatusScope Remote Monitoring Service User Monitoring Service to Guide perform these tasks: + Log on to the SCIEX Now™ account, + Search for a mass spectrometer Reference [How to contact Technical | Submit a support request: = ‘Support for software sciex.com/request-support support andtoleam about|, Telephone: (877) 740-2128 optional software (toll-free, North America only), ‘or go to sciex.com/contact-us to find a local office, Reference |How to contact the + Telephone: (877) 740-2129 = Technical Assistance (toll-free, North America only), Center (TAC) for or go to sciex.com/contact-us hardware support to find a local office. Reference |About warranty coverage|+ SCIEX website at = SCIEX warranties do not | Sciex-com/warranty cover removal, reinstallation, transportation of products, routine services such as cleaning, optimization, or connection to peripheral ‘components not ordered through SCIEX. Customer Familiarization Checklist for 3600, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 24183 RUO-IDV: 8-6776-HMethod [Topic Resource Complete | NIA Reference |About service products |» SCIEX website at sciex com. = and offerings included in | _ Navigate to the Support page the installation and and then click Instrument contract Service and Support. Reference |How to contact Customer|+ Submit a support request = Care to request a service| — sciex.com/request-support eal + Telephone: (877) 740-2129 (tolkfree, North America only), or go to sciex.comicontact-us to find a local office. Reference |About the Analyst > Release Notes for the Analyst = Diagnostic Tool Diagnostic Tool at sciex.com. Introduction to LC-MS/MS Operation RUO-IDV-08- 6776-4 Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems 25183Signoff 2 Note: The FSE adds the data and the completed electronic copy of the Customer Familiarization Checklist to the Files section of the Service Installation Case, for SCIEX intemal archival use. Organization Fischer Analytics GmbH FSE Name ‘Signature Date (yyyy-mm-dd) RSchneid 2021-07-28, Customer we have participated in Customer Familiarization and completed © all of the tasks identified in this checklist. Iwe have chosen to opt out of Customer Familiarization O° Name ‘Signature Date (yyyy-mm-dd) Name Signature Date (yyyy-mm-dd) Name ‘Signature Date (yyyy-mm-dd) Customers retain the signed hard copy for their records. Customer Familiarization Checklist for 3500, 4500, 5500, 5500+, and 6500+ Series Systems 26183 Introduction to LC-MS/MS OperationSignoff Comments If hard copies are required, then print only up to and including this page. The remainder of this document contains detailed instructions, and can be referenced electronically. NIA Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 27183Required Materials A Materials Supplied with the Purchase of a New System Part Number [Description Size ‘Quantity 4406127 |LC-MS Standards Kit with N/A 4 High/Low PPGs, 4376887 __ | Triazine standard solution N/A 1 4376878 [Phenomenex Synergi 4 um 50 mm x 2.0 mm 1 Fusion-RP 80 ALC column 4425163 [PEEK tubing (0.13 mm x 3000 mm 1 (0.008 inch i.d, 118 inches) 4403063 _|Finger-tight fitting (FTG), 10-pack 4/16 inch 1 weo10615 | Syringe tmb 1 1005819 [Syringe needle 1mL 1 1008236 [Syringe adapter N/A 1 4407334 LC vial, amber, 100-pack 2mL 1 4407331 LC blue screw cap with septa, N/A 1 100-pack Customer Familiarization Checklist for 3500, 4500, 5500, Introduction to LE-MS/MS Operation 5500+, and 6500+ Series Systems 28/83Required Materials Customer-Supplied Materials Descri Size Quantity Glass bottle, rinsed thoroughly to meet. 100 mL 4 standards of use with mass spectrometry Glass bottle, rinsed thoroughly to meet 4L 2 standards of use with mass spectrometry Beaker, rinsed thoroughly to meet 250 mL 2 standards of use with mass spectrometry Pre-blended LC-MS-grade methanol with 2.5L 1 0.1% formic acid’ Pre-blended LC-MS-grade water with 0.1% 25L 4 formic acid’ Customer-Supplied Lab Equipment Descri Size Quantity Adjustable micropipettes 400 yl and 1 mL 2 recommended Boxes of pipette 400 pL and 4 mL 2 recommended Measuring cylinder 100 mL. 2 Vortex mixer (optional) NIA 1 Glass transfer pipette NIA 1 PEEK tubing cutter NIA 1 1 pre-blende (LC-MS-grade) solvents not avaiable, then use the procedure in Altemative Solvent Preparation Method to prepare the solvent, Introduction to LC-MS/MS Operation RUO-IDV-08-6776-H Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems 29/83Hands-On Lab Exercise on LC-MS/MS Operation B Note: Before printing and using this procedure, make sure that the customer has completed all four of the modules in the Introduction to LC-MS/MS Operation Series for SCIEX Triple Quadrupole and QTRAP Systems eLeaming. This procedure accompanies the eLearning modules, and is intended to be used during the initial training on basic LC-MS/MS operations that is provided after instrument installation This hands-on lab exercise provides an example workflow for the typical tasks in LC-MS/MS operation. Users might need to customize the settings for a specific application. The following tasks are applicable to both the method developer workflow and the routine analysis workflow, unless otherwise specified. Note: Figures shown in this section are examples only. Actual screen content will vary depending on the LC system in use at the customer site, Preparation Guidelines + Use new glass bottles or bottles that are reserved for mobile phases only. + Use only LC-MS or higher grade solvents to wash or rinse the bottles. + Never use an injector waste bottle to prepare solvents. + Do not use tap water or unbottled Dl water. Prepare the Mobile Phases Using Pre-Blended Solvents WARNING! Toxic Chemical Hazard. Use a fume hood when pouring solvents or evaporating solvents. Refer to the chemical product Safety Data Sheets and follow all of the recommended safety procedures when handling, storing, and disposing of chemicals. Customer Familiarization Checklist for 3500, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 30/83Hands-On Lab Exercise on LC-MS/MS Operation Note: If pre-blended LC-MS-grade methanol with 0.1% formic acid or LC-MS-grade water with 0.1% formic acid is not available, then follow the steps in Alternative Solvent Preparation Method. Required Materials + Mobile phase A: Pre-blended LC-MS-grade water with 0.1% formic acid + Mobile phase B: Pre-blended LC-MS-grade methanol with 0.1% formic acid + Triazine standard solution Label the mobile phase bottles. Rinse the 1 L bottle for mobile phase A using mobile phase A, Fill the mobile phase A bottle with mobile phase A. Rinse the 1 L bottle for mobile phase B using mobile phase B, Fill the mobile phase B bottle with mobile phase B. akene Prepare the Wash Solvent for the Autosampler and Syringe Cleaning Required Materials + Mobile phase A + Mobile phase B + 100 mL bottle + 100 mL graduated cylinder 4. Use aclean 100 mL graduated cylinder to measure 75 mL of mobile phase B and 25 mL of mobile phase A, and then add them to a clean 100 mL bottle. 2. Mix the solvent well Label the solvent as Autosampler Wash Solvent and then set it aside for use with the autosampler. Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 31/83Hands-On Lab Exercise on LC-MS/MS Operation Prepare the Triazine Dilutions Required Materials + Mobile phase A + Mobile phase B + Triazine standard solution + 100 mL clean glass bottle + 2ml glass autosampler vials + 1 mL micropipette + 100 iL micropipette + (Optional) Vortex mixer 1. To prepare the triazine diluent, add 100 mL of mobile phase A to a 100 mL clean glass bottle and then label the bottle Triazine diluent. 2. Open the triazine standard solution by snapping the top of the vial Transfer the solution to a 2 mL glass autosampler vial 4. Prepare 1 autosampler vials, labeling the first one 1000 ng/mL. For the labeling of the final concentrations of the other vials, refer to Table B-1 Use a 1 mL micropipette to add 990 iL of dilution solvent to the first vial 6. Use a 100 iL micropipette to add 10 pL of triazine standard solution. Close the vial cap and then mix with a vortex mixer or by shaking vigorously. This is the 1000 ng/mL stock solution. 7. Starting with the 1000 ng/mL stock solution, perform serial dilutions by spiking the previous solution to the next one. The spiking amount is shown in the Volume of Starting Solution column in the following table. Mix each vial with a vortex mixer or by shaking it vigorously. Table B-4 Triazine Concentrations Starting Volume of Starting | Volume of Final Concentration Concentration Solution (ul) (ul) (ng/mL) - Record on (ng/mL) Vial Label 1000 20 980 20 20 500 500 10 10 200 800 2 Customer Familiarization Checklist for 3500, 4500, 5500, Introduction to LE-MS/MS Operation 5500+, and 6500+ Series Systems 32/83Hands-On Lab Exercise on LC-MS/MS Operation Table B-1 Triazine Concentrations (continued) Starting Volume of Starting ] Volume of Diluent [Final Concentration Concentration Solution (uL) (ul) (ng/mL) - Record on (ng/mL) Vial Label 2 500 500 1 1 200 800 02 02 500 500 04 Blank 0 4,000 Blank 8. Close the autosampler vial caps. Note: The diluted triazine can be stored at ambient temperature for up to three months. Prepare the Infusion Dilution for Optimization (Method Developer Workflow) Required Materials + Mobile phase A + 250 mL clean glass beaker + 2ml glass autosampler vials + 1 mL micropipette + 100 uL micropipette + (Optional) Vortex mixer 1. Pour approximately 20 mL of mobile phase A into a thoroughly clean glass beaker, 2, For 3500 series systems, follow these steps. a. Inaclean2 ml glass autosampler vial, add 900 iL of mobile phase A from the beaker. b. Add 100 pL of the 1,000 ng/ml triazine standard solution to the vial, to create the 100 ng/mL. solution. ©. Mix each vial with a vortex mixer or by shaking it vigorously. 3. For 4500, 5500, 5500+, or 6500+ series systems, follow these steps: a. Inaclean2 ml glass autosampler vial, add 990 j1L of mobile phase A from the beaker. Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 33/83Hands-On Lab Exercise on LC-MS/MS Operation b, Add 10 ul of the 1,000 ng/mL triazine standard solution created above to the vial, to create the 1 ng/mL solution . Mix the vial with a vortex mixer or by shaking it vigorously. Change the Mobile Phases 6. Put the mobile phase A line in the mobile phase A bottle (water with 0.1% formic acid), Put the mobile phase B line in the mobile phase B bottle (methanol with 0.1% formic acid). Purge the LC pumps. Refer to the documentation that comes with the LC system. For ExionLC AC Pumps, ExionLC AD Pumps, and Shimadzu pumps, open the purge valves on the LC pumps before purging. Pour approximately 20 mL of autosampler wash solvent in a clean glass beaker. The solvent is used to rinse the syringe in the following procedure. Put the wash solvent line in the autosampler wash solvent bottle. Note: If there is more than one wash line, then put all of the lines in the wash solvent bottle Purge the autosampler rinse line. Refer to the documentation that comes with the LC system. Configure the System for Infusion Connect the needle to the 1 mL syringe, 2, Rinse the syringe with the wash solvent from the beaker at least three times. 3. Fill the syringe with the triazine infusion dilution, as specified in the following table, making sure that all of the air bubbles are removed from the syringe Table B-2 Triazine Concentrations ‘System Concentration 3500 Systems 100 ng/mL 4500, 5500, 5500+, or 6500+ systems 410 ng/mL. Note: For dilution instructions, refer to Prepare the Infusion Dilution for Optimization (Method Developer Workflow). 4, Remove the needle and then connect the syringe to the PEEK infusion line. Customer Familiarization Checklist for 3500, 4500, 5500, Introduction to LE-MS/MS Operation 5500+, and 6500+ Series Systems 34183Hands-On Lab Exercise on LC-MS/MS Operation Install the syringe in the syringe pump. (Turbo V"/lonDrive™ Turbo V lon Source) Connect the infusion line to the ESI probe on the ion source. {OptiFlow Turbo V lon Source) Connect the infusion adapter to the top of the ion source and the line to the MICRO 1-50 iL or MICRO 50-200 HL probe, whichever is installed, (Turbo VilonDrive Turbo V lon Source) Verify the micrometer settings for the ESI probe: + Vertical: 5 + Horizontal: § Optimize for the Compound (Method Developer Workflow) 1. Create a project by following these steps a. Click Tools > Project > Create Project. b. Type the Project name. For example, Installation Training ©. Click OK. 2. Activate the hardware profile by following these steps: a. On the Navigation bar, under Configure, double-click Hardware Configur The Hardware Configuration Editor opens b. Select the Mass Spec Only profile appropriate to the system and then click Edit Profile. ©. Select the Mass Spectrometer device, and then click Setup Device. Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3500, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 35/83Hands-On Lab Exercise on LC-MS/MS Operation 4d. e f. Make sure that Use integrated syringe pump is selected, and then click OK, igure B-1 Use Integrated Syringe Pump — Srcmen ae "She Sharam eee Sextet Sees Flusnayassmaanvane ate [Mienayactwrerne)| io re Click OK to retum to the Hardware Configuration Editor. Click Activate Profile, Note: To create a new hardware profile, refer to the System User Guide for the mass spectrometer. 3. Start the syringe pump by following these steps: a. b. Select Tune and Calibrate mode. On the Navigation bar, under Tune and Calibrate, double-click Manual Tuning, The Tune Method Editor opens. Select Syringe Pump Method Set the Flow Rate to 10 and the Syringe Diameter to 4.61, if 1.0 mL syringe is used Click Start Syringe Pump. Click MS Method in the method list. Customer Familiarization Checklist for 3600, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 36/83 RUO-IDV: 6776Hands-On Lab Exer ise on LC-MS/MS Operation g. On the MS tab, set the parameters as specified in the following table. Table B-3 MS Parameters Field Value ‘Scan Type (Qt MS (Q1) Start (Da) 200 Stop (Da) 300 Duration (min) 3 Time (sec)* 1 Scan rate (Dals)* 1,000 * Ifavailable. These fields are not available for all instruments. h. Click Start i. Wait until the signal is stable. Tip! If the previous or expected ion chromatogram pane is hidden, then select the window to be shown from the dow menu, Note: Make sure that the intensity of the compound of interest is between 1e6 and 2e6, If it is higher, then dilute the triazine solution 10 times with the diluent. j. Close the Tune Method Editor without saving the method, The Instrument Settings dialog opens. (On the Navigation bar, under Tune and Calibrate, double-click Compound Optimization. 5. Configure the instrument settings by following these steps: a. Inthe Inlet area, select Infusion. b. Inthe Mass Spectrometer area, select MS/MS Analysis cc. Click Next, The lons to use in MS/MS Analysis dialog opens. 6, Configure the ions to be used by following these steps: a. Inthe Precursor lon area, select MW Ion, and then set the Search Range to + 0.6 Da. b. Inthe Polarity area, select Positive. c. Inthe Product lon area, select User Specified. Introduction to LC-MS/MS Operation RUO-IDV-08- 6776-4 Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems 37183Hands-On Lab Exercise on LC-MS/MS Operation d. Click Next The Target Components dialog opens. e. Identify the target components as shown in the following table, and then click Finish to begin the optimization process. Table B-4 Target Compounds Compound Name Q1 Mass (Da) Q3 Mass (Da) [Ametryn 4 228 186 ‘Ametryn 2 228 68 Atrazine 1 216 174 Atrazine 2 216 104 Prometon 1 226 142 Prometon 2 226 184 The screen shows two panes, a text file pane and an acquisition pane. It might be necessary to minimize one pane to see the other. The experiment being run is shown on the top of the acquisition pane, The X-axis shows the parameter that is being optimized for each experiment. The text file window is updated as results are generated. After optimization is complete, an MRM acquisition file is created and named compound_QOpt_FinalMRM_Pos.dam, where compound is the first compound in the Target Compound page. 7. After the compound optimization is completed, make sure that the Reserve Instrument for Tuning (| ~ ) button is not pressed, to allow acquisition. Customer Familiarization Checklist for 3500, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 38/83Hands-On Lab Exercise on LC-MS/MS Operation Plumb the LC System and Load the Samples Note: If the LC system was not installed by a SCIEX FSE at the time of the mass spectrometer installation, then the following sections will not be covered during the installation training: + Create the Acquisition Method (Method Developer Workflow) + Create the Acquisition Batch + Submit the Batch If this is the case, then use the sample data files downloaded from the fourth module within the Introduction to LC-MS/MS Operation Series for SCIEX Triple Quadrupole and QTRAP Systems eLeaming, 1, Turbo V/lonDrive Turbo V lon Source) Install the column: a, Connect the autosampler outlet tubing to the column inlet. b, Connect the PEEK tubing from the column outlet to the ion source inlet, 2. Load samples in an autosampler rack as specified in the following table and then carefully install the rack in the autosampler. Table B-5 Sample Vial Positions Sample | Blank | 0.005 | 0.01 0.05 04 0.5 | 4ngimL Name ngimt | ngimt | ngimt | ngimL | ngimt Vial 1 2 3 4 5 6 7 Position Turbo V/lonDrive Turbo V lon Source) Adjust the vertical micrometer setting to 2. 4, (OptiF low Turbo V Ion Source) Follow these steps: a. Connect the column to the probe, b, Connect the sample tubing between the column and the LC system. c. Install the column heater on the OptiFlow Turbo V lon Source. Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 39/83Hands-On Lab Exercise on LC-MS/MS Operation Acquisition Create the Acquisition Method (Method Developer Workflow) 1, Onthe Navigation bar, under Configure, double-click Hardware Configuration, deactivate the Mass Spec Only profile, activate the LCMS profile, and then click Close. 2 Click Acquire. Open the MRM methods created during compound optimization. Refer to Optimize for the Compound (Method Developer Workflow). 4, Select the MRM method and add IDs as shown in the following figure. Do not change the other optimized values. Set the dwell time to 40 ms for all transitions. Figure B-2 MRM Table 21 Has (0a)] 23 tase (Da)] Time (msee)] IO] oP vote | CE vot) | CXP (ott) [ase _ asst a0 myn? [76000 [arom P7000 Z| 720-100 |sao00 Jan [Amstyn? [76000 [63 o00__| 10.000 3 |ie.100 [74.000 400 [Arazine.1 [77-000 [27.00 [16.000 fa_f26-00 [104.000 [40.0 Atrazine 2 /7-000 (43.000 [5.000 =| zs 100s. ow0 a0 Premston 7760008 ood [8000 =| 225.100 [4.200400 Promston2 76000 2700 | W008 z Note: For this example, make sure that the DP values are greater than 20, Set the Duration to 5 minutes. 6. Toedit the Source/Gas parameters, follow these steps’ a. Click Edit Parameters. b. Click the Source/Gas tab. Customer Familiarization Checklist for 3500, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 40/83Hands-On Lab Exercise on LC-MS/MS Operation . Set the fields on the Source/Gas dialog to the values shown in the following table. ‘Table B-6 Source/Gas Parameters for the lonDrive Turbo V and Turbo Vion Source Field Value Curtain Gas (CUR) 35 lonSpray Voltage (ISV) 3600 Temperature (TEM) 650 lon Source Gas 4 (GS1) 60 lon Source Gas 2 (GS2) 60 CAD Medium or 9, as applicable d. Click OK. Create an LC method. Refer to LC Methods for LC method setup for supported HPLC systems ‘Add the LC method by following these steps. a. Right-click Acqui Figure B-3 Add/Remove Device Method cr ont 000 Sor ae ets Dace arta coun renee ep BP 0 b. Select an LC system to add to the method. Introduction to LC-MS/MS Operation RUO-IDV-08-6776-H Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems 41183Hands-On Lab Exercise on LC-MS/MS Operation ©. Make sure that the Synchronization Mode is set to LC Sync. Note: In LC Sync mode, the mass spectrometer coordinates with the action of the LC system to make sure that data is acquired properly. d. Select the LC system and then define the gradient profile as specified in the following table. Table B-7 Gradient Profile Time Flow (L/min) %A %B 0.0 600 80 20 o4 600 80 20 03 600 30 70 1.0 600 10 90 25 600 10 90 2.6 600 80 20 5.0 (stop time) 600 80 20 fe. Set the flow rate to 0,6 mUmin. f. Set the injection volume as specified in the following table. Table B-8 Injection Volume Mass Spectrometer ‘3500, 4500, 5500, or 5500+ series systems 6500+ series systems g. Set the minimum pressure to 300 psi and the maximum pressure to 6000 psi h. Ifa column oven is present, then set the column temperature to 40 °C. 9. Save the method with an appropriate name. For example, Triazine Final Method. Equilibrate the System Note: For optimal column performance, run a minimum of two blanks before the sample runs. Customer Familiarization Checklist for 3500, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 42183ise on LC-MS/MS Operation 1 Click View Queue (“) to open the sample queue. 2 Click Equilibrate (*") Figure B-4 Equilibrate Dialog Foul AeitonMehod Tecne inden 5 Inethin| 7 Fee ae ert eee ee ete! fomldette epee! sulin “Taig ih dk devs compton ater sabi in Ga Geet ae 3. Inthe Acquisition Method field, select Triazine final.dam. Type 3 in the Time (Min.) field and then click OK, Make sure that the pressure is stable. Note: Always use an appropriate equilibration time for the application and column size. When the system is ready, the icons on the status bar turn green. The status bar is located in the bottom right comer of the Analyst Software window. Figure B-5 Status Bar idle ME Ready FY Ready Tip! Ifthe equilibration does not finish as expected, or ifthe system status does not change to Ready after the equilibration finishes, then make sure that the following conditions are true: + The activated hardware profile is appropriate for the acquisition method + The LC system is tured on + The LC system has correctly established communication with the software Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3500, 4500, 5500, RUO-IDV-08- 5500+, and 6500+ Series Systems 6776-4 43183Hands-On Lab Exercise on LC-MS/MS Operation Create the Acquisition Batch 1. Follow these steps to create the batch’ a. On the Navigation bar, under Acquire, double-click Build Acquisition Batch, b. Click Add Set. c. Click Add Samples. The Add Samples dialog opens. d. Inthe New Samples Number field, type 14. fe. Click OK. 2, Select the acquisition method created in the previous procedure. This is the method that will be used for all of the samples, Tip! If the Inj. Volume field contains 1, then an acquisition method is not selected, the acquisition method is not configured for the current LC system, or the hardware profile does not match the current hardware configuration 3. Fillin the sample information, Refer to Figure B-7: a. Inthe Sample Name column, type the names of the samples. Note: The samples in vials 2, 4 and 6 are also used as QC Low, QC Medium, and QC High. The sample in vial 5 is also used as Unknown sample. b. Inthe Rack Code column, select the rack code. Figure B-6 Rack Code List iient fm cose Dees Wel > as oo ¢._ In the Vial position column, type the vial position of each sample. d. In the Data File column, rename the file, if required. Customer Familiarization Checklist for 3600, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 44183 RUO-IDV: 8-6776-Hise on LC-MS/MS Operation Tip! Use the fill-down function by selecting the column header, right-clicking, and then choosing Fill Down. Figure B-7 Samples Sump ame] fark Code] fuck oaibon]| Pate Cade| Pte Poston] Vl Poston] —TwinFie —[aVoume Wi] fae Frsmcosvit Hise Tosa Tr ian ang T038 afore — [isms [ise 15 [4 z insite za] 1.090 3 ozngint —isnctosvit [se 15 4 x insite sang] 1.00 get [iscsi [se 15 4 Te ian vai T03 3 [zag isn osvit ak [t iE ian vai T03 forgo [rane esi ak [t = insiten sang] 1.00 Fang [sacs 0 TF sttton vag] 10.00 ror sac tos a0 i alton vig 10098 3 [otiee snes a0 z alton vig] 10098 a ES a0 Te alton vig] 10098 i rE a0 Te alton vig] 10098 Tz [ere snc tos¥it a0 i alton vig] 10098 a brows sa tos it a0 5 alton vig] 10098 7a [are PSR tosvit ies 1 i alton rig] 1008 4. Before submitting samples, use Quick Quant to add compound concentrations. Follow these steps Tip! Quick Quantis recommended for the method developer workflow, and not for routine analysis. The method developer should create a complete quantitation method. Refer to Data Processing (MultiQuant Software) or Data Processing (SCIEX OS). a. Click Quick Quant Figure B-8 Quick Quant Button cuansten cs Note: The method created with Quick Quant is a semi-quantitation method. It is used only to migrate the sample concentrations to the data processing software. The Create Semi-Automatic Quantitation Method dialog opens. b. Click OK. . Type a file name for the quantitation method. For example, QuickQuant. d. Click OK. Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 45/83Hands-On Lab Exercise on LC-MS/MS Operation e. Click the Locations tab and then double-click the blank space inside the tab to show the autosampler rack layout. Figure B-9 Locations Tab (Before and After) O} Ss ‘S| S S ‘2, oF o (=) Ss O} orarO-O-OO- OOO OO OB 00.0000000000000 gy JODOOOO OOD OO DOO 00000000 = S o o S S S S S S o 3 = 3 5 o o o 5 o S S S S S o Ss 0,050 f. Click the Quantitation tab. g. Inthe Quant Type column, assign the correct sample type to each sample. Customer Familiarization Checklist for 3500, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 46/83 RUOADV-08-6776-HHands-On Lab Exercit ¢ on LC-MS/MS Operation 5, h. In the concentration field, type the concentration of the samples for the first transition, and then copy (Ctrl+C) the concentration and paste it (Ctrl+V) in the other columns. Figure B-10 Quantitation Tab ‘Sample | Lecatons| Guerttnen | Sut TL) AertMead — atatontainng nace grt ae [eat a Toarocoo [cocoon —[ococo —ootenea —[atooo 20. gmt —[Sindied [01 1.0000) — [0 ‘omc0 —[o.sene0 —[o 0000 soz maint [sindae [02 1.209000 — [0.20000 —|o-00n00 — [0 20000 fe] rege [Sind [1 rocco — | reoace —| +0060 — [1 aeooo fe] grt——[Sindad |? econ) —[zeoato —|zonenco — [2 ato [5] 10 min [Standd [10 To 060 |W 00000 [0 cooo | 000 | rom. [sind [20 20.0000 — [20.0000 — 20 on000 [21 como fears foareoe0 —|aceoo00 — [oeooce —|oouone0 —|oaene0o fa —lacter Jac of 1.00000 [ato —|o.re000 [0-100 0] 9C med la ft otcno0 —| 109900 — | a00009 — |. 90000 tf aC gh Ta ra i oonot —[oonc00 — |v on00o —[teooae [Sak Sik fe aroc0o —[nceco00 —[o.coonce —|ooonea —[o cane [a |Urknorn [Unknown [6 af0000 — [0.000001 —[conomo —|oonen00 — [0 nt000 Fa Tata tak foar0000 — To de0000 — to eamca To ommee Ta neo Save the batch with the name Installation training batch. Submit the Batch Note: For optimal column performance, perform system equilibration before starting the batch, and then make sure that the pressure is stable. 1, Click the Submit tab, Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 47183Hands-On Lab Exercise on LC-MS/MS Operation ‘Shoo [Lactos |Suntinon| 500" ach Done nae Use Sa tm alc ares ard ean Daal User Integration Defaults. Configure the global settings that will apply to all of the analytes Tip! To change the regression parameters, click Edit > User Units and Calibration Defaults. Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 491.83Hands-On Lab Exercise on LC-MS/MS Operation Figure B-12 Integration Defaults Dialog seg te TT] nussan Smet Wcth: [5 points xno [nin srtenvanaowe BO see upastessecean [ho (> Report Lzest Pes sun.reukatn: [poms in. roukneunt: [207 — INoserercentaze: [00 _« 0K. conse! Figure 8-13 Units & Calibration Defaults Coreetnton nis gE ——— Repent: new =] —— | x cnc Click File > New Quantitation Method. 4. Select a mi level concentration sample (the 1 ng/mL sample), and then click OK. In the Components table, type the compound name in the Group text box. Note: If internal standards are being used, they would be identified as IS and assigned to specific analytes. For information about the Intemal Standards menu, refer to the. MultiQuant Software Reference Guide. Customer Familiarization Checklist for 3600, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 50/83 RUO-IDV-08-6776-HHands-On Lab Exercise on LC-MS/MS Operation Click the Integration & Regression tab. If necessary, change the default processing Parameters for individual analytes by typing in the new value, and then clicking Apply Click the Outlier Settings tab and then make sure that Accuracy for Standards and Accuracy for QCs are selected, and that the values are set as shown in the following figure. Figure B-14 Outlier Settings (Compormts| ogaion Fegeonon OU Serge a IF Acusoy fr Sande fecuray fer Cs Mex tocmncyTaeerestr U0a ovens [TH % || Max heey Tdementr cc: [TS Max AocumcyTdernce fr Ses cep L0G: [—F5 % Click Save. The Save As dialog opens Type a file name. For example, "Installation training_triazine quant’ Click Save. The quantitation method is ready for use. Create a Results Table 1. Click File > New Results Table. The Select Samples dialog opens. 2. Select the samples by following these steps: a. Browse to the data file folder. b. Move the entire data file to the Selected pane ©. Click Next 3. Choose the method by following these steps: a. Under Choose existing method, click Open. b. Select the triazine quantitation method created in the previous section. ©. Click Open. d. Click Finish The Results Table is created. 4. To save the Results Table, click File > Save As Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3500, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 51/83Hands-On Lab Exercise on LC-MS/MS Operation 5. Type a file name. For example, “Install training results", 6. Cick Save. Review the Results Table Tip! The results can be filtered to show All Components, Groups, or individual transitions, To change the view, select one of the options listed on the left side of the Results Table. 1. Compare the Actual Concentration column to the sample sheet used to prepare the standards and QCs to make sure that there is no mismatch of the expected concentrations. Tip! To change a value, type the new value in the cell. 2, Click the transition prometon.1 in the Components and Groups pane. Figure B-15 Components & Groups Pane (Q Mattiquant Gi Fite Edit Process Audit & Br BW) Girstalation tr [All Components |amatryn Group smetryn Group ‘atrazine Group 3. If required, select the columns to be shown in the Results Table by following these steps. a. Right click inside the Results Table. Customer Familiarization Checklist for 3600, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 52/83 RUO-IDV: 6776Hands-On Lab Exercise on LC-MS/MS Operation b. Click Column Settings Figure B-16 Right-Click Menu (Column Settings. ‘Ada Custom Column. Rename Custom Column, Remove Custom Column ‘Apply Curent Analyte's Actual Concentrations to All ‘Apply Curent S's Actual Concentrations to All Set Usea! , ‘Set Peaks 19'Not Found for Selacted Rows c. Select the Visible check box beside each column to be shown, For example, select the Visible check box beside Conc. Units to show this column in the Results Table. Figure B-17 Column Settings = Joanne d. Click OK. Tip! The customized settings can be exported for future use. They can also be saved as the default settings, Introduction to LC-MS/MS Oper Customer Familiarization Checklist for 3500, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-8776-H 53/83Hands-On Lab Exercise on LC-MS/MS Operation 4, Review the peak integration by following these steps: a. Click Displays the peak review (EB) to show the chromatograms. b. To inspect the shape of a peak, zoom in. c. To reintegrate a peak, drag the mouse over the peak The integration is automatically applied Tip! Alternatively, change the integration parameters and then click Apply to update the peak integration Tip! To change the appearance of the Peak Review pane, right-click and then click Options. The layout of the pane can be changed in the following ways: + The number of rows and columns shown + Whether the internal standard is overlaid with the analytes. + The amount of zoom for the peaks. Note: A manual integration option is also available. Manually integrate peaks only if all the peaks have not been found after the algorithm parameters have been adjusted and updated. We recommend that users validate the results to determine whether manual integration is acceptable for specific applications, Figure B-18 Manual Integration sr) d, To delete the pane, click Deletes this pane (I), Note: This step deletes the pane from the workspace, but it does not delete the data Tip! To sort or filter the list, use the tools in the Result Table toolbar. Refer to the MultiQuant Software Reference Guide. 5. To review the calibration curve, click Displays the calibration curve (Id The calibration curve is shown. The regression equation, the r value, and the weighting factor are also shown, Customer Familiarization Checklist for 3500, 4500, 5500, Introduction to LE-MS/MS Operation 5500+, and 6500+ Series Systems 54183ise on LC-MS/MS Operation Tip! If the accuracy of the calibration point is not within acceptable limits, then right-click a data point within the pane and click Exclude. This can also be accomplished by clearing the box in the Used column in the Results Table. RUO-IDV-08- (Method developer workflow) Change the regression setting a. Click Regression. The regression options are shown b. Set the appropriate regression and weighting for each compound Note: Routine analysis users should not change the regression settings. They should notify the method developer or lab manager if changes are required to improve calibration. 7. Todelete the pane, click Deletes this pane (| 8 Click Displays the statistics pane. Figure B-20 Displays the statistics pane Icon 8. Select Group by concentration for standards and then select one of the options. For example, select Group by concentration for QCs to show the accuracy and precision for quality controls. With this function, the user can review the standard and QC results. 10. Repeat step 4 to step 9 to review the calibration curve, peak integration, and statistics for each analyte, using the Components and Groups pane to select the analytes. Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3500, 4500, 5500, 5500+, and 6500+ Series Systems 6776-4 55/83Hands-On Lab Exercise on LC-MS/MS Operation Note: The user should always review peak integration 11, To review the metric plot for the retention time, click the Retention Time column in the 2). The metric plot can be used to identify variations in retention time, peak area, and so on, for both analytes and internal standards. 12. _ Review the Results Table and verify that all of the changes have been made, Then, save the Results Table Results Table and then click Create a Metric Plot 13. Create a report by following these steps: Click File > Create Report and Save Results Table. b. Select a report template from the list. For example, Calibration Curves Template . Click Set, browse to the location where the report will be saved, and then type a name for the report. For example, Install training report. Figure B-21 Create Report Dialog {Pik Create Report eae a Rosertensss: [CsibGres Tomei dosr——~—S~*~*~*~*S*SY penn et om = TF Generate alae in Hack ad it OupFomat: — C word C HTML @ FOF © CSV TF Crene an ini rpor fr each sale (econrende fo large reper) ox d. Set the output format to Word, HTML, POF, or CSV. e. Click OK. 14. _ Review the report by using File Explorer to navigate to the location in which the report was saved, and then opening the report Data Processing (SCIEX OS) In this section, the user creates a processing method based on the AutoPeak algorithm. Two other algorithms are available, the MQ4 algorithm and the Summation algorithm. For more information about the algorithms, refer to the Software User Guide for SCIEX OS, Customer Familiarization Checklist for 3600, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 56/83 RUO-IDV: 8-6776-HHands-On Lab Exercise on LC-MS/MS Operation Build a Processing Method (Method Developer Workflow) 1, Onthe SCIEX OS Status panel, make sure that the project that contains the triazine data is the active project. 2. Open the Analytics workspace. Click Projects > Project Default Settings 4. Configure the quantitative processing parameters as shown in the following figure. These settings will apply to all of the analytes. Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 57/83Hands-On Lab Exercise on LC-MS/MS Operation Figure B-22 Quantitative Processing Parameters ees ‘Set reject wide deta for quantitative procsting mated parameters Qualitative Processing Method te, Mass Reconstruction Processing Srucamnaiens [pereeey| imgitontigatomn [Aah ¥] Wh recent s009 a o —_ “Miscou peak bight [000] vernsminen [aw] agen oe eet © Click Save and then click Close. Click Process Method > New. In the Available list, select all of the samples in the batch, and then click the right arrow to move the samples to the Selected box, Customer Familiarization Checklist for 3600, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 58/83 RUOADV-08-6776-HHands-On Lab Exercit ¢ on LC-MS/MS Operation 8, Click Components and then confirm all of the components and transitions. If a group was not assigned automatically, then typea group name for each pair of components that belongs to the same compound. 9. Configure the integration parameters: a. Click Integration. b. Click Calculate method parameters. ¢. For one component, select Remove outliers automatically from the calibration curve. d. Type ng/mL for Concentration units. e, Select Apply units and calibration parameters to all of the components, f. Click Apply. 10. _ Review the integration for all components. If necessary, change the processing parameters for individual analytes by typing the new value, and then clicking Appl. 11. Configure the flagging rules’ a. Click Flagging Rules Figure B-23 Modify Method ——— New. 2. Select the data file from the Available list, and then click the right arrow to move the entire data file to the Selected list 3, Click the Browse button under Select a Processing Method and then browse to the triazine processing method created in the previous procedure. 4. Click Open to select the processing method. Make sure that Update Integration parameters based on currently selected samples is selected. Figure B-26 Select a Processing Method eee es ©) Upaitebtegaton parenetes based once alee sone 6. Click Process. The Results Table is created. 7. Click Results > Save As. 8 Browse to the location of the folder that contains the triazine data, and then select the Quantitation Results folder. 9. Type a file name. For example, “Install training results", 10. Click Save, Review the Results Table Tip! The results can be shown by individual samples, by All Components, by Groups, or by individual transitions. To change the view, select the Samples or Components and Groups tab to the left of the Results Table. 1, Compare the Actual Concentration column to the sample sheet used to prepare the standards and QCs to make sure that there is no mismatch of the expected concentrations. Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 61/83Hands-On Lab Exercise on LC-MS/MS Operation Tip! To change a value, type the new value in the cell. 2, Click the transition Prometon 2 in the Components and Groups pane. 3, To customize the columns to be shown in the Results Table, follow these steps a. Click More > Table display settings. b, Select the required columns, and then click OK, Make sure that the following columns are visible: + Accuracy + Accuracy Acceptance + lon Ratio Confidence + Integration Acceptance For example, select lon Ratio Confidence to show the ion ratio traffic lights in the Results Table, Tip! The customized settings can be exported for future use. They can also be saved as the default settings. Select Save as project default settings to keep the Results Table display settings. 4. Tofilter the results shown in the Results Table, follow these steps a. Click the filter icon on any column header. For example, select the filter icon on the Sample Type column. Figure B-27 Filter Icon brows raver: 0 BA) Quatty for Rules Index| Sample Name | Sampe..©) Component. ©) Compound.. Customer Familiarization Checklist for 3600, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 62/83 RUO-IDV: 8-6776-HHands-On Lab Exercit ¢ on LC-MS/MS Operation b. Clear any values to be excluded from the Results Table, For example, clear Blank and Double Blank. Figure B-28 Sample Type Filter 1b tron tere 0 |B Quay for al anual Ramil Peak Review. Tip! Alternatively, click Displays the Peak Review (Ell) b, To inspect the shape of a peak, left-click and then drag outside of the X- or Y-axis to zoom the peak display. ©. To reintegrate a peak, drag the cursor over the peak within the graph The integration is automatically applied. Tip! Alternatively, change the integration parameters and then click Apply to update the peak integration Tip! To change the appearance of the Peak Review pane, click Options > Peak review display settings. The layout of the pane can be changed in the following ways: + The number of rows and columns shown. + Whether the internal standard is overlaid with the analytes, + The amount of zoom for the peaks. Note: A manual integration option is also available. Manually integrate peaks only if none of the peaks have been found after the algorithm parameters have been adjusted and updated. We recommend that users validate the results to determine whether manual integration is acceptable for specific applications. d. Click OK. e. To close the Peak Review pane, click the * in the top corner of the pane. Note: This step deletes the pane from the workspace, but it does not delete the data Customer Familiarization Checklist for 3500, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 64/83Hands-On Lab Exercit ¢ on LC-MS/MS Operation 7. To review the calibration curve, click Views > Calibration Curve. Tip! Alternatively, click Displays the Calibration Curve (2) The calibration curve is shown. The regression equation, the r value, and the weighting factor are also shown. Tip! If accuracy of the calibration point is not within acceptable limits, right-click a data point within the pane and then click Exclude selected analyte. Alternatively, exclude the analyte by clearing the check box in the Used column in the Results Table. 8. (Method developer workflow) Change the regression setting a. Click Regression. The regression options are shown. b. Ifrequired, set the appropriate regression and weighting for each compound. Note: Routine analysis users should not change the regression settings, They should notify the method developer or laboratory manager if changes are required to improve calibration 8. To close the Calibration Curve pane, click the in the top comer of the pane 10. To review the statistics, follow these steps Click Views > Statistics pane b. Select Group by concentration for standards and then select one of the options. For example, select Group by concentration for QCs to show the accuracy and precision for quality controls. With this function, the user can review the standard and QC results ©. Tochange the statistics value types, such as Area or Calculated Concentration, select the option from the list. d. To close the Statistics pane, click the * in the top comer of the pane. 11, Repeat step 6 to step 10 to review the calibration curve, peak integration, and statistics for each analyte, using the Components and Groups pane to select the analytes Note: The user should always review peak integration. 12. To review the metric plot for the retention time, click the Retention Time column in the Results Table and then click More > Create Metric Plot with new settings. Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3500, 4500, 5500, ‘8500+, and 6500+ Series Systems RUO-DV-06-6776-4 65/83Hands-On Lab Exercise on LC-MS/MS Operation The metric plot can be used to identify variations in retention time, peak area, and so on, for both analytes and internal standards. 13. (Optional) To make the metric plot easier to review, customize its appearance by selecting options from the Options menu. 14, Review the Results Table and verify that all required changes have been made, 15. Create a report by following these steps a. Click Reporting > Create report and save Results Table. b. Select a report template from the list. For example, Calibration Curves, ©. Set the output format to Word, HTML, PDF, or CSV. Tip! The CSV format does not apply to the report template graphics. d. (Optional) Click Browse. e. Browse to the location where the report will be saved, and then type a name for the report. For example, "Install training report”. Then click Save. f Click Create The report automatically opens in the selected format. 16. _ Review and then close the report. Customer Familiarization Checklist for $600, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 66/83 RUO-IDV: 8-6776-HLC Methods Cc Note: Figures are examples only. Actual screen content will vary depending on the version of the software and the model of the LC modules installed. ExionLC Systems Figure C-1 Pump Properties Injection BB] eH] AD Pump @ren ene sire foe [0] mura Se ER es Go $00 o«m0a e009 aad oriram [20 Maviwm | E000 Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3500, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-8776-H 67/83LC Methods Figure C-2 Autosampler Properties fa AD Autosampler Dhecietin cate ee noe nT i sero s 35] war coat tree sot 3 1 Gna eryerre sl Seen ae —— $9 sown ato 2 os ee hr ot on =. a on ve see ae emai orennne i an ricejor —Pastowihy [169 wn Cente ange ge Figure C-3 Column Oven Properties FL 40 column oven cao oe a = : oven ampere: a) {2 i for tempaatre ulbraon before un 3 wonewene EBs oe | ~ ‘Heat compensation few resin pore ‘Customer Familiarization Checklist for 3500, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 68/83 RUO-IDV-08-676-HLC Methods Figure C-4 Controller Properties tena aonpuge ore wes (D Tenpemp nate avoouge Introduction to LC-MS/MS Oper Customer Familiarization Checklist for 3500, 4500, 5500, 5500+, and 6500+ Ser RUO-IDV-08-8776-HLC Methods Agilent 1260 and 1290 Systems Figure C-5 LC Pump Gradient Properties. LC Pump Siac [Lins [insides Mi Mode trom | many | AC | BO f a aan Ci Figure C-6 Limits Prope LeFune Gada] Lins [Lin advance) | Mim Mode) Parsnece Mir Prac 3 {sm00 ourum Pieri [roo stake okee fo) MoirunFbRonpUs (une?) [1000 MasruinFbeRlanpDown lie) 00 Customer Familiarization Checklist for 3500, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 70/83LC Methods Figure C-7 Agilent Autosampler Properties (3500, 4500, 5500, and 5500+ Series Systems) Tlenatorenge Popeter [advanced Peeeee] ibe Coss ‘waehDaal Siege Sis fa Ensbes Ircoionveimetih [300 ‘at Leos: Drow Sreedhvmint figoo Wash el -829s201 [5 Ect Speed wid) [aon WathCdes(1-5) Fy NecdoLevelirm [oo Wahid urbe Wah Pack Number [7 “Tenperotre canto! Dena Saban -0C, Figure C-8 Agilent Autosampler Properties (6500+ Series Systems) ‘ager rorohr Preven | Bdvareed epee Inet tate Wath Dette Srge Soe a Dienaties econ Vole FF Wath locaton: [EGBA Draw Seced umn 080 Wash Tro (1-889900% [F fet Soe ilee) o Wash cites 0-3: Nese Levin): [0 Wet il arbor Jeoeauecoes Wash Ack rhinber ‘aronilé-A0C} Sp Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 71183LC Methods Figure C-9 Agi nt Column Oven Properties Salen Calm ivenPene ee Terps Let Teepatue| Fig TerpeatuerC) Teper Tobe +-(C} Sat qin Toes +) Coben Shing Ve © Use sate coer fr Customer Familiarization Checklist for 3600, 4500, 5500, 5500+, and 6500+ Series Systems 72183 RUO- D Introduction to LC-MS/MS Operation 08-676-HLC Methods Agilent 1260 and 1290 Infinity Il Systems Figure C-10 Pump Properties [iy wren Canales er Fre dire Ce a) rows: rd [ert Phan Seep me Pit sero oo nares \ or = Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 73183LC Methods Figure C-12 Column Compartment Properties File vew fiony fume Matisamper Colin Core Temper let Fike O Nexconatit © Nerconatics ® a0} ® pos] © se detec Cet © Ae dee Cat © Unchanged © Unchanged © Combines Customer Familiarization Checklist for 3600, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 74183 RUOADV-08-6776-HLC Methods Shimadzu Systems Figure C-13 Pumps Properties Pumps |Aulosamplet] Oven | Gankollet | Time Program| Pumping Mode: [Binary Flow TotaiFlow 08 myn PumpE Cone 20 % Pumpecuve 0 Configuied Pumps Pump: L¢-20AD Pump. LC-208D Pump: none Pump D: none Piovsuie Limits (PumpA.B) Miu: 300 si Maximum: 6000, pai Introduction to LC-MS/MS Operation RUO-IDV-08-6776-H Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems 75183LC Methods Figure C-14 Autosampler Properties Pumps) Auosampter [oven. | Gonsole | Time Program) Mose ILDORGXR neck ype Undetned (Decarea] Wise Autosampler Rinsing Volume, 20 uw Needle Stoke 52 mm Rinsing Speed: % uLfsoe SompingSpee 5 ubpsec Puge Tine 250 rin Rinse Dip Time: 0 sec Rinse Mode (Beto andafieraspiaion WeEnable Cooler Unit Cooler Temperatire: 15 c Convol Vial Needle Stoke: 52 mm Figure C-15 Oven Properties Pumps [Awosampler) Oven Mods [OTO-20AC WEnable Oven Oven Temperature: (40 c Maximum Temperature: [85 ce Customer Familiarization Checklist for 3600, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 76183 RUOADV-08-6776-HLC Methods Figure C-16 Time Program Properties Time Program [Time |Medule | Event [Parmeter i TT Pumps Pump 8 Conc. a0 2 03 Pumps Puma 8 Conc. 70 a 10 Pumps Pump Gane 0 4 25 Pumps Pump 8 Conc 0 5 28 Pumps Puma B Conc. 0 a 50 Contollr Stop z J Graken Cuve +109 a o oi ® af 00 10 20 30 40 50 Time (in) Figure C-17 Injection Volume (3500, 4500, 5500, and 5500+ Series Systems) Acristion method Autosameler EM! Acquisition Method -@, Mass Spec 5.000 mit Inection Vue fu} fo ©-G Period 5.000 mir 8 -vRM edd Shimadzu LC Syste at Equilbrate Injection Customer Familiarization Checklist for 3500, 4500, 5500, Introduction to LC-MS/MS Oper 5500+, and 6500+ Ser RUO-IDV-08-8776-HLC Methods Figure C-18 Injection Volume (6500+ Series Systems) Acqusiton method Aulosamler TRY Acquistion Method eG, Mass Spec 5.000 min Iriecion Vou [u) BG Period 5,000min $8 +1vRM A shimadeu Lc system & Equilbcate +o Customer Familiarization Checklist for 3600, 4500, 5500, Introduction to LC-MS/MS Operation 5500+, and 6500+ Series Systems 78183 RUO-IDV: 8-6776-HAdditional Resources D This section contains additional resources that the customer can refer to after the FSE-led familiarization is complete. Hardware Topic Resource Vacuum system configuration: + Roughing pump + Turbo pump + Vacuum (system) shutdown + Vacuum pump-down (system startup) + SCIEX University”: Customer Familiarization + SCIEX University”: Front End Cleaning of ‘SCIEX Triple Quadrupole and QTRAP’ ‘Systems + System User Guide for the mass spectrometer Software This section lists resources with information about the Analyst Software components that are used to control the LC-MS/MS system, Operating System Software Topic Resource ‘About the Analyst Data folder and Project structure + SCIEX University™: Analyst’ Software Overview + Laboratory Director Guide + Analyst Software Help Introduction to LC-MS/MS Operation RUO-IDV-08-6776-H Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems 79183Additional Resources Configure Mode Note: For more information about configuring SCIEX OS, refer to the Laboratory Director Guide and Software User Guide for SCIEX OS. Topic Resource How to configure Analyst Software security + Security modes: + Integrated mode (default) + Mixed mode (Cliquid Software) + Single User mode + Project security + Activate and deactivate Laboratory Director Guide, included in the Analyst Software documentation set, Refer to Documentation, Support, and Training (10 min). How to add users. Laboratory Director Guide Analyst Software Help How to configure the LC-MS/MS system hardware + Mass spectrometer only + Add a diverter valve + Add a syringe pump + Add an LC system SCIEX University™: Analyst” Software Overview ‘System User Guide for the mass spectrometer The User Guide or Operator Guide for the LC system. How to plumb the diverter valve in injector mode and diverter mode System User Guide for the mass spectrometer Projects and subprojects + Create a project + Create a subproject ‘SCIEX University™: Analyst’ Software Overview ‘System User Guide for the mass spectrometer Analyst Software Help Customer Familiarization Checklist for 3500, 4500, 5500, 5500+, and 6500+ Series Systems 80/83 Introduction to LC-MS/MS Operationional Resources Tune and Calibrate Mode Topic Resource How to optimize ion source parameters for stable spray (in high and low flow) ‘Manual Compound Optimization Tutorial ‘System User Guide for the mass spectrometer Analyst Software Help Quantitate Mode We recommend the use of SCIEX OS or MultiQi Juant Software for quantitative analysis. Refer to the SCIEX OS Software User Guide or MultiQuant Software Reference Guide. The Quantitate Mode of the Analyst Software can also be used for quantitative analysis, as well as for qualitative analysis. For descriptions of the workflows, refer to the System User Guide for the mass spectrometer. Topic Resource Build a Quantitation Method (method developer workflow) ‘System User Guide for the mass spectrometer Create a Results Table (method developer and routine analysis workflows) ‘System User Guide for the mass spectrometer Review a Results Table (method developer and routine analysis workflows): Review peak integration + Review a calibration curve + Review statistic results Review a metric plot Save the results table with a new name ‘System User Guide for the mass spectrometer Reporter Software Topic Resource Create a report with the Reporter Software ‘System User Guide for the mass spectrometer Introduction to LC-MS/MS Operation RUO-IDV-08-6776-H Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems 81/83Addit nal Resources Maintenance Front-End Full Cleaning Topic Resource How to clean the orifice plate while the mass spectrometer is vented (following the customer cleaning procedure) + SCIEX University”: Front End Cleaning of SCIEX Triple Quadrupole and QTRAP" ‘Systems + Instrument Front-End Cleaning Procedure, available at sciex.com How to clean the QJet/lonDrive™ QJet lon Guide (following the customer cleaning procedure) Instrument Front-End Cleaning Procedure, available at sciex.com How to clean the Q0 rod set (following the customer cleaning procedure) Instrument Front-End Cleaning Procedure, available at sciex.com Customer Familiarization Checklist for 3600, 4500, 5500, 5500+, and 6500+ Series Systems 82/83 Introduction to LC-MS/MS Operation RUO-IDV-08-6776-HAlternative Solvent Preparation Method E WARNING! Toxic Chemical Hazard. Use a fume hood when pouring solvents or evaporating solvents. Refer to the chemical product Safety Data Sheets and follow all of the recommended safety procedures when handling, storing, and disposing of chemicals. Note: If pre-blended LC-MS-grade methanol with 0.1% formic acid or LC-MS-grade water with 0.1% formic acid is not being used, then follow these steps to prepare the mobile phases. Required Materials + LC-MS-grade water + LC-MS-grade methanol (stored in glass bottles) + Formic acid ampules, 99% or higher (2 x 1 mL) Label the mobile phase bottles. Rinse the 1 L bottle for the mobile phase A using the LC-MS-grade water. Rinse the 1 L bottle for the mobile phase B using the LC-MS-grade methanol. Rinse the 1 L graduated cylinder with LC-MS-grade water. Carefully measure 1 L of LC-MS-grade water and then transfer it to the labeled mobile phase A bottle. eeRena 2 Open a formic acid ampule and then pour all of the formic acid in the bottle. Mix the solvent well This is mobile phase A 8. Rinse the 1 L graduated cylinder with LC-MS-grade methanol 9. Carefully measure 1 L of LC-MS-grade methanol and then transfer it to the labeled mobile phase B bottle. 10. Open a second formic acid ampule and then pour all of the formic acid in the bottle. 11. Mix the solvent well, This is mobile phase B. Introduction to LC-MS/MS Operation Customer Familiarization Checklist for 3800, 4500, 5500, 5500+, and 6500+ Series Systems RUO-IDV-08-6776-H 83/83