5500 Series of Instruments

System User Guide

RUO-IDV-05-6768-A December 2017

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,
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Portions of this document may make reference to other manufacturers and/or their products, which may contain parts whose
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SCIEX warranties are limited to those express warranties provided at the time of sale or license of its products and are SCIEX’s
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For research use only. Not for use in diagnostic procedures.
AB Sciex is doing business as SCIEX.
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AB SCIEX™ is being used under license.
© 2017 AB Sciex

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Woodlands Central Indus. Estate.
SINGAPORE 739256

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Contents

Chapter 1 Operational Precautions and Limitations................................................................................8

General Safety Information................................................................................................................................................8
Regulatory Compliance......................................................................................................................................................8
Australia and New Zealand..........................................................................................................................................8
Canada........................................................................................................................................................................9
Europe.........................................................................................................................................................................9
United States...............................................................................................................................................................9
International..............................................................................................................................................................10
Electrical Precautions.......................................................................................................................................................10
AC Mains Supply........................................................................................................................................................10
Protective Earth Conductor........................................................................................................................................11
Chemical Precautions.......................................................................................................................................................11
System Safe Fluids.....................................................................................................................................................12
Ventilation Precautions....................................................................................................................................................13
Environmental Precautions..............................................................................................................................................14
Electromagnetic Environment....................................................................................................................................14
Decommissioning and Disposal..................................................................................................................................15
Qualified Personnel..........................................................................................................................................................16
Laboratory Conditions......................................................................................................................................................16
Operating Conditions.................................................................................................................................................16
Performance Specifications........................................................................................................................................16
Equipment Use and Modification.....................................................................................................................................17
Contact Us.......................................................................................................................................................................17
Technical Support............................................................................................................................................................18
Documentation Symbols and Conventions.......................................................................................................................18
Related Documentation...................................................................................................................................................19
Chapter 2 Principles of Operation............................................................................................................20
System Overview..............................................................................................................................................................20
Hardware Overview...................................................................................................................................................21
Theory of Operation—Hardware ..............................................................................................................................24
®
Analyst Software Overview .....................................................................................................................................26
®
Theory of Operation—Analyst Software..................................................................................................................33
Chapter 3 Operating Instructions — Sample Workflows.......................................................................36
Chapter 4 Operating Instructions—Hardware........................................................................................45
Start Up the System.........................................................................................................................................................45
Reset the Mass Spectrometer...........................................................................................................................................46
Shut Down and Vent the System......................................................................................................................................46

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Contents

Adjust the Integrated Syringe Pump Position...................................................................................................................47

Plumb the Diverter Valve.................................................................................................................................................51
Plumb the Diverter Valve in Injector Mode................................................................................................................52
Plumb the Diverter Valve in Diverter Mode................................................................................................................53
Chapter 5 Operating Instructions — Hardware Profiles and Projects...................................................55
Hardware Profiles............................................................................................................................................................55
Create a Hardware Profile..........................................................................................................................................55
Add Devices to a Hardware Profile............................................................................................................................59
Troubleshoot Hardware Profile Activation.................................................................................................................61
Projects and Subprojects..................................................................................................................................................62
Create Projects and Subprojects.................................................................................................................................62
Create Subprojects.....................................................................................................................................................63
Copy Subprojects.......................................................................................................................................................63
Switch Between Projects and Subprojects..................................................................................................................64
Installed Project Folders.............................................................................................................................................64
Back Up the API Instrument Folder............................................................................................................................65
Recover the API Instrument Folder.............................................................................................................................65
Chapter 6 Tune and Calibrate..................................................................................................................66
Verify Instrument Performance........................................................................................................................................66
About the Verifying or Adjusting Performance Dialog.....................................................................................................67
Results Summary..............................................................................................................................................................67
Chapter 7 Automatic Optimization..........................................................................................................69
About Automatic Optimization........................................................................................................................................70
Types of Sample Introduction.....................................................................................................................................70
Automatically Optimize for an Analyte Using Infusion.....................................................................................................71
Confirm the Presence of Compounds.........................................................................................................................71
Perform Automatic MS and MS/MS Optimization Using Infusion with a
Known Precursor Ion and an Unknown Product Ion..................................................................................................73
Review the Optimization Results...............................................................................................................................76
Automatically Optimize for an Analyte Using FIA............................................................................................................77
Chapter 8 Operating Instructions — Acquisition Methods....................................................................84
Create an Acquisition Method Using the Acquisition Method Editor...............................................................................84
Configure the Integrated Syringe Pump.....................................................................................................................85
Add an Experiment....................................................................................................................................................85
Add a Period..............................................................................................................................................................86
Copy an Experiment into a Period..............................................................................................................................86
Copy an Experiment within a Period..........................................................................................................................86
Scan Techniques..............................................................................................................................................................87
Quadrupole-Mode Scan Types...................................................................................................................................87
LIT-Mode Scan Types.................................................................................................................................................88
About Spectral Data Acquisition......................................................................................................................................89
Chapter 9 Operating Instructions — Analyze and Process
Quantitative Data.....................................................................................................................................90
Quantitative Analysis.......................................................................................................................................................90
Quantitation Methods................................................................................................................................................90

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About Results Tables.................................................................................................................................................91

Quantitation Methods and Results Tables.................................................................................................................91
Create a Method Using the Quantitation Method Editor...........................................................................................91
Create a Results Table Using the Quantitation Wizard..............................................................................................93
Create a Standard Query (Optional)...........................................................................................................................94
Results Table Right-click Menu..................................................................................................................................97
Peak Review and Manual Integration of Peaks................................................................................................................98
Review Peaks.............................................................................................................................................................98
Manually Integrate Peaks........................................................................................................................................103
Peak Review Right-Click Menu................................................................................................................................104
Calibration Curves.........................................................................................................................................................105
View Calibration Curves...........................................................................................................................................105
Overlay Calibration Curves.......................................................................................................................................107
Calibration Curve Right-Click Menu.........................................................................................................................107
Sample Statistics............................................................................................................................................................108
View the Statistics for Standards and QCs...............................................................................................................108
Compare Results Between Batches................................................................................................................................109
Chapter 10 Operating Instructions — Analyze and Explore Data.......................................................110
Open Data Files..............................................................................................................................................................110
Navigate Between Samples in a Data File......................................................................................................................110
View Experimental Conditions.......................................................................................................................................111
Show Data in Tables......................................................................................................................................................112
Show ADC Data.............................................................................................................................................................114
Show Basic Quantitative Data........................................................................................................................................115
Chromatograms.............................................................................................................................................................116
Show TICs from a Spectrum...........................................................................................................................................117
Show a Spectrum from a TIC..........................................................................................................................................117
About Generating XICs..................................................................................................................................................118
Generate an XIC Using a Selected Range.................................................................................................................119
Generate an XIC Using the Maximum Peak.............................................................................................................119
Generate an XIC Using Base Peak Masses...............................................................................................................120
Extract Ion by Selecting Masses...............................................................................................................................120
Generate BPCs...............................................................................................................................................................121
Generate XWCs..............................................................................................................................................................123
Generate DAD Data.......................................................................................................................................................123
Generate TWCs..............................................................................................................................................................124
Adjust the Threshold......................................................................................................................................................124
Chromatogram Panes....................................................................................................................................................125
Spectra Panes................................................................................................................................................................126
Graphical Data Processing.............................................................................................................................................126
Graphs...........................................................................................................................................................................127
Manage Data...........................................................................................................................................................127
Zoom In on the Y-axis..............................................................................................................................................129
Zoom In on the X-axis..............................................................................................................................................129
Chapter 11 Reporter Software...............................................................................................................130
Analyst Reporter User Interface.....................................................................................................................................131

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Contents

Generate Reports...........................................................................................................................................................133
Chapter 12 Operating Instructions — Batches......................................................................................135
Set Queue Options.........................................................................................................................................................135
Create and Submit a Batch............................................................................................................................................136
Add Sets and Samples to a Batch............................................................................................................................136
Equilibrate the System.............................................................................................................................................139
Submit a Sample or Set of Samples..........................................................................................................................140
Change Sample Order....................................................................................................................................................140
Acquire Data..................................................................................................................................................................140
Set Sample Locations in the Batch Editor.......................................................................................................................141
Select Vial Positions Using the Locations Tab (Optional)...............................................................................................142
Set Quantitation Details in the Batch Editor (Optional)..................................................................................................143
Stop Sample Acquisition................................................................................................................................................143
Import Batch Files..........................................................................................................................................................144
Build a Batch as a Text File......................................................................................................................................144
Import a Batch from a Text File................................................................................................................................144
Batch Editor Right-click Menu........................................................................................................................................145
Queue States and Device Status....................................................................................................................................146
Queue States............................................................................................................................................................146
View Instrument and Device Status Icons................................................................................................................147
Queue Right-click Menu...........................................................................................................................................148
Chapter 13 Service and Maintenance Information...............................................................................150
Recommended Maintenance Schedule..........................................................................................................................150
Clean the Surfaces.........................................................................................................................................................152
Clean the Front-End.......................................................................................................................................................152
Symptoms of Contamination....................................................................................................................................153
Required Materials ..................................................................................................................................................153
Cleaning Best Practices............................................................................................................................................154
Prepare the Mass Spectrometer...............................................................................................................................155
Clean the Curtain Plate............................................................................................................................................156
Clean the Front of the Orifice Plate..........................................................................................................................157
Put the Mass Spectrometer Back in Service.............................................................................................................157
Inspect the Roughing Pump Oil Level............................................................................................................................158
Empty the Source Exhaust Drain Bottle..........................................................................................................................158
Storage and Handling....................................................................................................................................................160
Chapter 14 Mass Spectrometer Troubleshooting.................................................................................161
Appendix A Operating Instructions — Manual Compound Optimization..........................................164
About Manual Compound Optimization........................................................................................................................164
About Scan Types...........................................................................................................................................................165
Manually Optimize an Analyte.......................................................................................................................................165
Confirm the Presence of Compounds.......................................................................................................................165
Optimize MS-Specific Parameters............................................................................................................................167
Determine the Product Ions for Optimization...........................................................................................................168
Optimize Collision Cell Exit Potential for each Product Ion......................................................................................170
Manually Optimize the Ion Source and Gas Parameters................................................................................................171

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Prepare the Ion Source.............................................................................................................................................171

Optimize the Ion Source Parameters........................................................................................................................171
Advanced Parameters....................................................................................................................................................172
Optimize AF2...........................................................................................................................................................172
About Collision Energy Spread (CES) ......................................................................................................................173
Appendix B 5500 Series System Parameters.........................................................................................174
Appendix C Calibration Ions and Solutions..........................................................................................177
Appendix D Toolbar Icons......................................................................................................................180
Appendix E Glossary of Symbols...........................................................................................................189
Appendix F Glossary of Warnings.........................................................................................................194

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Operational Precautions and
Limitations 1
Note: Before operating the system, carefully read all of the sections of this guide.

This section contains general safety-related information and provides regulatory compliance information. It also
describes potential hazards and associated warnings for the system and the precautions that should be taken to
minimize the hazards.
In addition to this section, refer to Glossary of Symbols on page 189 for information about the symbols and
conventions used in the laboratory environment, on the system, and in this documentation. Refer to the Site
Planning Guide for site requirements, including AC mains supply, source exhaust, ventilation, compressed air,
nitrogen, and roughing pump requirements.

General Safety Information

To prevent personal injury or system damage, read, understand, and obey all of the safety precautions and warnings
in this document, the manufacturer chemical safety data sheet (SDS), and product label information. These labels
are shown with internationally recognized symbols. Failure to heed these warnings could result in serious injury.
This safety information is intended to supplement federal, state, provincial, and local environmental health and
safety (EHS) regulations. The information provided covers system-related safety information applicable to the
operation of the system. It does not cover every safety procedure that should be practised. Ultimately, the user
and the organization are responsible for compliance with federal, state, provincial, and local EHS regulations and
for maintaining a safe laboratory environment.
Refer to the appropriate laboratory reference material and standard operating procedures.

Regulatory Compliance
This system complies with the regulations and standards listed in this section. Refer to the Declaration of Conformity
included with the system and the individual system components for dated references. Applicable labels have been
affixed to the system.

Australia and New Zealand

• Electromagnetic Compatibility (EMC): Radio Communications Act 1992 as implemented in these
standards:
• Electromagnetic Interference—AS/NZS CISPR 11/ EN 55011/ CISPR 11 (Class A). Refer to Electromagnetic
Interference on page 15.

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 Operational Precautions and Limitations

• Safety: AS/NZ 61010-1 and IEC 61010-2-061

Canada
• Electromagnetic Interference (EMI): CAN/CSA CISPR11. This ISM device complies with Canadian
ICES-001. Refer to Electromagnetic Interference on page 15.
• Safety:
• CAN/CSA C22.2 No. 61010-1
• CAN/CSA C22.2 No 61010-2-061

Europe
• Electromagnetic Compatibility (EMC): Electromagnetic Compatibility directive 2014/30/EU as
implemented in these standards:
• EN 61326-1
• EN 55011 (Class A)
Refer to Electromagnetic Compatibility on page 14.
• Safety: Low Voltage Directives 2014/35/EU as implemented in these standards:
• EN 61010-1
• EN 61010-2-061
• Waste Electrical and Electronic Equipment (WEEE): Waste Electrical and Electronic Equipment 2012/
96/EEC, as implemented in EN 40519. Refer to Waste Electrical and Electronic Equipment on page 16.
• Packaging and Packaging Waste (PPW): Packaging and Packaging Waste Directive 94/62/EC
• RoHS Restriction of Hazardous Substances: RoHS Directive 2011/65/EU

United States
• Radio Emissions Interference Regulations: 47 CFR 15, as implemented in FCC Part 15 (Class A)
• Safety: Occupational Safety and Health Regulations, 29 CFR 1910, as implemented in these standards:
• UL 61010-1
• IEC 61010-2-061

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Operational Precautions and Limitations

International
• Electromagnetic Compatibility (EMC):
• IEC 61326-1
• IEC CISPR 11 (Class A)
• IEC 61000-3-2
• IEC 61000-3-3
Refer to Electromagnetic Compatibility on page 14.
• Safety:
• IEC 61010-1
• IEC 61010-2-061

Electrical Precautions
WARNING! Electrical Shock Hazard. Do not remove the covers. Removing the
covers might cause injury or malfunctioning of the system. The covers need
not be removed for routine maintenance, inspection, or adjustment. Contact
a SCIEX Field Service Employee (FSE) for repairs that require the covers to be
removed.

• Follow required electrical safe work practices.

• Use cable management practices to control electrical cables. This will reduce the chance of a tripping hazard.
For information about system electrical specifications, refer to the Site Planning Guide.

AC Mains Supply
Connect the system to a compatible AC mains supply as instructed in this guide.

WARNING! Electrical Shock Hazard. Use only qualified personnel for the
installation of all of the electrical supplies and fixtures, and make sure that
all of the installations adhere to local regulations and safety standards.

WARNING! Electrical Shock Hazard. Make sure that the system can be
disconnected from the mains supply outlet in an emergency. Do not block
the mains supply outlet.

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 Operational Precautions and Limitations

WARNING! Electrical Shock Hazard. Use only the power cables supplied with
the system. Do not use power cables that are not properly rated for the
operation of this system.

An external line transformer is not needed for the mass spectrometer, optional bench, or roughing pump.

Protective Earth Conductor

The mains supply must include a correctly installed protective earth conductor. The protective earth conductor
must be installed or checked by a qualified electrician before the system is connected.

WARNING! Electrical Shock Hazard. Do not intentionally interrupt the

protective earth conductor. Any interruption of the protective earth conductor
creates an electrical shock hazard.

WARNING! Electrical Shock Hazard. Make sure that a protective earth

conductor (grounding cable) is connected between the sample loop and an
appropriate grounding point at the ion source. This supplementary grounding
will reinforce the safety configuration specified by SCIEX.

Chemical Precautions
WARNING! Radiation Hazard, Biohazard, or Toxic Chemical Hazard. Determine
whether decontamination is required prior to cleaning or maintenance. The
customer must decontaminate the system prior to cleaning or maintenance
if radioactive materials, biological agents, or toxic chemicals have been used
with the system.

WARNING! Environmental Hazard. Do not dispose of system components in municipal

waste. Follow local regulations when disposing of components.

WARNING! Biohazard, Toxic Chemical Hazard. Connect the drain tubing to

the mass spectrometer and the source exhaust drain bottle properly, to
prevent leaks.

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Operational Precautions and Limitations

• Determine which chemicals have been used in the system prior to service and regular maintenance. Refer to
the Safety Data Sheets for the health and safety precautions that must be followed with chemicals. SCIEX
Safety Data Sheets can be found at sciex.com/tech-regulatory.
• Work in a well-ventilated area or fume hood.
• Always wear assigned personal protective equipment, including powder-free neoprene or nitrile gloves, safety
glasses, and a laboratory coat.
• Avoid ignition sources when working with flammable materials, such as isopropanol, methanol, and other
flammable solvents.
• Take care in the use and disposal of any chemicals. Potential risk of personal injury if proper procedures for
handling and disposing of chemicals are not followed.
• Avoid skin contact with chemicals during cleaning and wash hands after use.
• Make sure that all exhaust hoses are connected properly and that all connections are functioning as designed.
• Collect all spent liquids and dispose of them as hazardous waste.
• Comply with all of the local regulations for the storage, handling, and disposal of biohazardous, toxic, or
radioactive materials.
• (Recommended) Use secondary containment trays beneath the roughing pump, the solvent bottles, and the
waste collection container to capture potential chemical spills.

System Safe Fluids

The following fluids can safely be used with the system. Refer to Required Materials on page 153 for information
about safe cleaning solutions.

CAUTION: Potential System Damage. Do not use any other fluid until confirmation is
received from SCIEX that it does not present a hazard. This is not an exhaustive list.

• Organic Solvents
• MS-grade acetonitrile; up to 100%
• MS-grade methanol; up to 100%
• Isopropanol; up to 100%
• HPLC-grade or higher water; up to 100%
• Buffers
• Ammonium acetate; less than 1%
• Ammonium formate; less than 1%

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 Operational Precautions and Limitations

• Acids and Bases

• Formic acid; less than 1%
• Acetic acid; less than 1%
• Trifluoroacetic acid (TFA); less than 1%
• Heptafluorobutyric acid (HFBA); less than 1%
• Ammonia/ammonium hydroxide; less than 1%

Ventilation Precautions
The venting of fumes and disposal of waste must comply with all of the federal, state, provincial, and local health
and safety regulations. It is the responsibility of the customer to make sure that the air quality is maintained in
compliance with local health and safety regulations.
The source exhaust system and roughing pump must be vented to a dedicated laboratory fume hood or an external
exhaust system.

WARNING! Fire Hazard. Make sure that the source exhaust system is connected and
functioning to prevent flammable vapor from accumulating in the ion source.

WARNING! Radiation Hazard, Biohazard, or Toxic Chemical Hazard. Take care

to vent exhaust gases to a dedicated laboratory fume hood or exhaust system
and make sure that the ventilation tubing is secured with clamps. Make sure
that the laboratory has appropriate air exchange for the work performed.

WARNING! Radiation Hazard, Biohazard, or Toxic Chemical Hazard. Do not

operate the mass spectrometer if the source exhaust drain and roughing
pump exhaust hoses are not properly connected to the laboratory ventilation
system. Perform a regular check of the exhaust tubing to make sure that
there are no leaks. The use of mass spectrometers without proper system
ventilation might constitute a health hazard and might result in serious injury.

WARNING! Radiation Hazard, Biohazard, or Toxic Chemical Hazard. Use the

ion source only if you have knowledge of and training in the proper use,
containment, and evacuation of toxic or injurious materials used with the
ion source.

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Operational Precautions and Limitations

WARNING! Puncture Hazard, Radiation Hazard, Biohazard, or Toxic Chemical

Hazard. Discontinue use of the ion source if the ion source window is cracked
or broken and then contact a SCIEX Field Service Employee (FSE). Any toxic
or injurious materials introduced into the equipment will be present in the
source exhaust output. Dispose of sharps following established laboratory
safety procedures.

Environmental Precautions
Use qualified personnel for the installation of electrical mains, heating, ventilation, and plumbing supplies and
fixtures. Make sure that all of the installations comply with local bylaws and biohazard regulations. For information
about the required environmental conditions for the system, refer to the Site Planning Guide.
Allow access space around the equipment when setting up the system.

DANGER! Explosion Hazard. Do not operate the system in an environment containing

explosive gases. The system is not designed for operation in an explosive
environment.

WARNING! Biohazard. For biohazardous material use, always comply with local
regulations for hazard assessment, control, and handling. This system or any part
is not intended to act as a biological containment.

CAUTION: Potential Mass Shift. Maintain a stable ambient temperature. If the temperature
changes by more than 2 °C per hour, then the resolution and mass calibration might be
affected.

Electromagnetic Environment
Electromagnetic Compatibility
Basic Electromagnetic Environment: Environment existing at locations characterized by being supplied
directly at low voltage from the public mains network.
Performance Criteria A (Criteria A): Equipment shall operate as intended with no degradation of performance
and no loss of function during or after test.
Performance Criteria B (Criteria B): Equipment may experience loss of function (one or more) during test
but shall operate as intended with some degradation of performance and functions self-recoverable after test.

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 Operational Precautions and Limitations

Performance Criteria C (Criteria C): Equipment may experience loss of function (one or more) during test
but shall operate as intended with some degradation of performance and functions recoverable by operator after
test.
The equipment is intended for use in a basic electromagnetic environment.
The expected performance loss under the electromagnetic immunity conditions is less than 60% change in total
ion count (TIC).
Make sure that a compatible electromagnetic environment for the equipment can be maintained so that the device
will perform as intended. If the power supply line is subject to high electrical noise, then install a surge protector.

Electromagnetic Interference
Class A Equipment: Equipment which is suitable for use in all establishments other than domestic and those
directly connected to a low voltage power supply network which supplies buildings used for domestic purposes.
[Derived from CISPR 11:2009, 5.3] Class A equipment shall meet Class A limits.
This equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to Part
15 of the FCC (Federal Communications Commission) Compliance Rules.
These limits are designed to provide reasonable protection against harmful interference when the equipment is
operated in a commercial environment. This equipment generates, uses, and can radiate radio frequency energy
and, if not installed and used in accordance with the operator's manual, can cause harmful interference to radio
communications.
Operation of this equipment in a residential area is likely to cause harmful interference in which case you will be
required to correct the interference, at your own expense. Changes or modifications not expressly approved by
the manufacturer could void your authority to operate the equipment.

Decommissioning and Disposal

WARNING! Environmental Hazard. Do not dispose of system components in municipal

waste. Follow local regulations when disposing of components.

Before decommissioning, decontaminate the entire system following local regulations.

When removing the system from service, separate and recycle different materials according to national and local
environmental regulations. Refer to Storage and Handling on page 160.

Note: SCIEX will not accept any system returns without a completed Decontamination Form. Contact an FSE
to obtain a copy of the form.

Do not dispose of system components or subassemblies, including computer parts, as unsorted municipal waste.

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Operational Precautions and Limitations

Waste Electrical and Electronic Equipment

Follow local municipal waste ordinances for proper disposal provisions to reduce the environmental impact of
waste, electrical, and electronic equipment (WEEE). To safely dispose of this equipment, contact a local Customer
Service office for complimentary equipment pick-up and recycling.

Qualified Personnel
Only qualified SCIEX personnel shall install, inspect, and service the equipment. After installing the system, the
Field Service Employee (FSE) uses the Customer Familiarization Checklist to orient the customer on system
operation, cleaning, and basic maintenance.
Only personnel qualified by the manufacturer shall maintain the equipment. A laboratory designate can be
familiarized with the Qualified Maintenance Person (QMP) procedures during the installation. A QMP is a person
who is suitably aware of the electrical and chemical risks associated with servicing laboratory equipment.

Laboratory Conditions
Operating Conditions
The system is designed to operate safely under these conditions:
• Indoors
• Altitude: Up to 2 000 m (6 400 feet) above sea level
• Ambient temperature: 5 °C (41 °F) to 40 °C (104 °F)
• Relative humidity: 80% for temperatures up to 31 °C (88 °F), decreasing lineraly to 50% at 40 °C (104 °F)
• Mains supply voltage fluctuations: ±10% of the nominal voltage
• Transient overvoltages: Up to the levels of Overvoltage Category II
• Temporary overvoltages on the mains supply
• Pollution degree: Pollution Degree 2

Performance Specifications
The system is designed to meet specifications under these conditions:
• Ambient temperature: 15 °C (59 °F) to 30 °C (86 °F)
Over time, the temperature must remain within a range of 4 °C (7.2 °F), with the rate of the change in
temperature not exceeding 2 °C (3.6 °F) per hour. Ambient temperature fluctuations exceeding the limits might
result in mass shifts in spectra.
• Relative humidity: 20% to 80%, non-condensing

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 Operational Precautions and Limitations

Equipment Use and Modification

WARNING! Personal Injury Hazard. Contact the SCIEX representative if product
installation, adjustment, or relocation is required.

WARNING! Electrical Shock Hazard. Do not remove the covers. Removing the
covers might cause injury or malfunctioning of the system. The covers need
not be removed for routine maintenance, inspection, or adjustment. Contact
a SCIEX Field Service Employee (FSE) for repairs that require the covers to be
removed.

WARNING! Personal Injury Hazard. Use SCIEX-recommended parts only. Use of parts
not recommended by SCIEX or use of parts for any purpose other than their intended
purpose can put the user at risk of harm or negatively impact system performance.

Use the mass spectrometer and ion source indoors in a laboratory that complies with the environmental conditions
recommended in the Site Planning Guide.
If the mass spectrometer and ion source is used in an environment or in a manner not prescribed by the
manufacturer, then the protection provided by the equipment might be impaired.
Unauthorized modification or operation of the mass spectrometer and ion source might cause personal injury and
equipment damage, and might void the warranty. Erroneous data might be generated if the mass spectrometer
and ion source is operated either above or below the recommended environmental conditions or operated with
unauthorized modifications. Contact an FSE for information on servicing the system.

Contact Us
SCIEX Support
• sciex.com/contact-us
• sciex.com/request-support

Customer Training
• In North America: NA.CustomerTraining@sciex.com
• In Europe: Europe.CustomerTraining@sciex.com
• Outside the EU and North America, visit sciex.com/education for contact information.

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Operational Precautions and Limitations

Online Learning Center

• SCIEXUniversity

CyberSecurity
For the latest guidance on cybersecurity for SCIEX products, visit
sciex.com/Documents/brochures/win7-SecurityGuidance.pdf.

Technical Support
SCIEX and its representatives maintain a staff of fully-trained service and technical specialists located throughout
the world. They can answer questions about the system or any technical issues that might arise. For more
information, visit the SCIEX website at sciex.com.

Documentation Symbols and Conventions

The following symbols and conventions are used throughout the guide.

DANGER! Danger signifies an action which leads to severe injury or death.

WARNING! Warning signifies an action that could cause personal injury if precautions
are not followed.

CAUTION: Caution signifies an operation that could cause damage to the system or
corruption or loss of data if precautions are not followed.

Note: Note emphasizes significant information in a procedure or description.

Tip! Tip provides useful information that helps apply the techniques and procedures in the text for a specific
need and provides shortcuts, but is not essential to the completion of a procedure.

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 Operational Precautions and Limitations

Related Documentation
To find software product documentation, refer to the release notes or software installation guide that comes with
the software. Documentation for the hardware products can be found on the Customer Reference DVD that
comes with the system or component.
For the latest versions of the documentation, visit the SCIEX website at sciex.com.

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Principles of Operation 2
The 5500 system is designed for the qualitative and quantitative analysis of chemical species.
®
This section includes information about the mass spectrometer and the Analyst software. Refer to the ion source
Operator Guide for an overview of the ion source.
®
For information on the computer and software, refer to the Software Installation Guide for the Analyst
software.

WARNING! Lifting Hazard. Do not move the system. Risk of personal injury
or system damage. If the system must be moved, then contact a Field Service
Employee (FSE).

System Overview
WARNING! Lifting Hazard. Make sure that at least six people are available to lift
the mass spectrometer. Follow established safe lifting procedures. Refer to the Site
Planning Guide for the weights of system components.

This system is intended for the qualitative and quantitative analysis of chemical species.
The 5500 series of instruments includes the following components:
TM ®
• A SCIEX Triple Quad 5500 or QTRAP 5500 mass spectrometer with a roughing pump and a source of
compressed air and nitrogen
TM ®
• Turbo V ion source that uses either the TurboIonSpray probe or the atmospheric pressure chemical ionization
TM
(APCI) probe. Refer to the Turbo V Ion Source Operator Guide.
®
• SCIEX-supplied computer and monitor with the Analyst software for instrument optimization, acquisition
method development, processing, and data acquisition. For computer specifications and requirements, refer
®
to the Analyst Software Installation Guide.

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 Principles of Operation

Hardware Overview

Figure 2-1 Front View

Item Description Refer to...

1 Panel symbols Panel Symbols on page 21.
2 Syringe pump Adjust the Integrated Syringe Pump Position on page 47
TM
3 Ion source Turbo V Ion Source Operator Guide, available from the
ion source documentation DVD, or from the SCIEX Web site, at
sciex.com
4 Diverter valve Plumb the Diverter Valve on page 51.

Panel Symbols
Table 2-1 describes the mass spectrometer status LEDs.

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Principles of Operation

Table 2-1 Panel Symbols

LED Color Name Description

Green Power Lit when the system is powered up.

Green Vacuum Lit when the correct vacuum level has been
achieved. Flashing if the vacuum is not at the
correct level (during pump down and venting).
Green Ready Lit when the system is in the Ready state. The
system must be in the Ready state to operate.

Blue Scanning Flashing when the system is acquiring data.

Red Fault Lit when the system encounters a system fault.

After the system is turned on, all of the LEDs illuminate. The power LED remains lit. The other LEDs flash for two
seconds and then turn off. The vacuum LED starts flashing. After the correct vacuum level is achieved this LED
remains lit.

Connections
Figure 2-2 shows the location of the mass spectrometer connections, including the locations of the RESET and
VENT buttons and the mass spectrometer convenience switch.

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 Principles of Operation

Figure 2-2 Back and Side Views

Item Description Primary Materials For more information...

1 Roughing pump vacuum Aluminum (hose fitting), Contact an FSE.
connection zinc-plated steel (hose clamp)
2 Air supply (Gas 1/Gas 2) Plastic Refer to the Site Planning Guide.
Gas tubing from the Gas 1/Gas 2 supply
connects to this port.
3 Source exhaust supply Stainless steel Refer to the Site Planning Guide.
Gas tubing from the source exhaust gas
supply connects to this port.
4 Source communication Aluminum Contact an FSE.
connection
5 RESET button Plastic Refer to Reset the Mass Spectrometer
on page 46.

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Principles of Operation

Item Description Primary Materials For more information...

6 Mains supply connection Aluminum/plastic Refer to Start Up the System on page
45 or Shut Down and Vent the System
on page 46.
7 Mass spectrometer Plastic Refer to Start Up the System on page
convenience switch 45 or Shut Down and Vent the System
on page 46.
(Up = On; Down = Off)
8 Aux I/O connection Sheet metal (zinc-plated) Refer to the Peripheral Devices
Setup Guide.
9 Ethernet connection Sheet metal (zinc-plated) Contact an FSE.
(connects the mass
spectrometer and the
computer)
10 VENT button Plastic Refer to Start Up the System on page
45 or Shut Down and Vent the System
on page 46.
11 Source exhaust waste (to Stainless steel Refer to the Site Planning Guide.
waste bottle)
12 Nitrogen gas supply Stainless steel Refer to the Site Planning Guide.
TM
(Curtain Gas supply, Gas tubing from the nitrogen gas supply
CAD gas) connects to this port.

Theory of Operation—Hardware
Mass spectrometry measures the mass-to-charge ratio of ions to identify unknown compounds, to quantify known
compounds, and to provide information about the structural and chemical properties of molecules.
The mass spectrometer has a series of quadrupole filters that transmit ions according to their mass-to-charge
®
(m/z) ratio. The first quadrupole in this series is the QJet ion guide located between the orifice plate and the Q0
®
region. The QJet ion guide does not filter ions, but focuses them before they enter the Q0 region. By prefocusing
®
the larger ion flux created by the wider orifice, the QJet ion guide increases system sensitivity and improves the
signal-to-noise ratio. In the Q0 region, the ions are again focused before passing into the Q1 quadrupole.

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 Principles of Operation

Figure 2-3 Ion Path

Item Description
1 Orifice plate
2 QJet ion guide
3 Q0 region
4 Q1 quadrupole
5 Q2 collision cell
6 Q3 quadrupole
7 Detector

The Q1 quadrupole is a filtering quadrupole that sorts the ions before they enter the Q2 collision cell. The Q2
collision cell is where the internal energy of an ion is increased through collisions with gas molecules to the point
that molecular bonds break creating product ions. This technique allows users to design experiments that measure
the m/z of product ions to determine the composition of the parent ions.
After passing through the Q2 collision cell, the ions enter the Q3 quadrupole for additional filtering, and then they
enter the detector. In the detector, the ions create a current that is converted into a voltage pulse. The voltage
pulses leaving the detector are directly proportional to the quantity of ions entering the detector. The system
monitors these voltage pulses and then converts the information into a signal. The signal represents the ion
intensity for a particular m/z value and the system shows this information as a mass spectrum.
The linear ion trap (LIT) functionality provides a number of enhanced modes of operation. A common factor of the
enhanced modes is that ions are trapped in the Q3 quadrupole region and then scanned out to produce full
spectrum data. Many spectra are rapidly collected in a short period of time and are significantly more intense than
spectra collected in a comparable standard quadrupole mode of operation.

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Principles of Operation

During the collection phase, ions pass through the Q2 collision cell where the CAD gas focuses the ions into the
Q3 region. The Q3 quadrupole is operated with only the main RF voltage applied. Ions are prevented from passing
through the Q3 quadrupole rod set and are reflected back by an exit lens to which a DC barrier voltage is applied.
After the fill time elapses (a time defined by the user, or determined by the Dynamic Fill Time feature), a DC barrier
voltage is applied to a Q3 entrance lens (IQ3). This confines the collected ions in Q3 and stops additional ions
from entering. The entrance and exit lens DC voltage barriers and the RF voltage applied to the quadrupole rods
confine the ions within Q3.
During the scan out phase, the voltage on the exit lens and the auxiliary RF voltage are ramped simultaneously
with the main RF voltage for increased resolution and sensitivity as compared to quadrupole scan modes. An
auxiliary AC frequency is applied to the Q3 quadrupole. The main RF voltage amplitude is ramped from low to
high values, which sequentially brings masses into resonance with the auxiliary AC frequency. When ions are
brought into resonance with the AC frequency, they acquire enough axial velocity to overcome the exit lens barrier
and are axially ejected towards the mass spectrometer ion detector. Full spectra data can be acquired from the
ions collected in Q3 by rapidly scanning the main RF voltage.

Data Handling
®
The Analyst software requires a computer running the Windows 7 (32- or 64-bit) operating system or the Windows
10 (64-bit) operating system. The computer and the associated system software work with the system controller
and the associated firmware to control the system and data acquisition. During system operation, the acquired
®
data is sent to the Analyst software where it can be shown as either full mass spectra, intensity of single or
multiple ions over time, or total ion current over time.
®
Analyst Software Overview
®
The Analyst software works with the mass spectrometer and the liquid chromatography (LC) system and the
associated firmware to control the system and data acquisition. During system operation, the acquired data is
®
sent to the Analyst software where it can be shown as full mass spectra, intensity of single or multiple ions over
time, or total ion count over time.

Different Data Views

The following figures show examples of two types of data views: total ion chromatogram (TIC) and extracted ion
chromatogram (XIC).
TIC: The plot of the total ion current as a function of time.

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 Principles of Operation

Figure 2-4 Example TIC

XIC: An ion chromatogram created by taking intensity values at a single, discrete mass value or a mass range,
from a series of mass spectral scans. It indicates the behavior of a given mass or mass range as a function of time.

Figure 2-5 Example XIC

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Principles of Operation

Parameters
The working parameters are the set of instrument parameters currently being used.
• Source and gas parameters: These parameters can change depending on the ion source used.
• Compound parameters: These parameters consist mostly of voltages in the ion path. Optimal values for
compound-dependent parameters vary depending on the compound being analyzed.
• Resolution parameters: These parameters affect the resolution and calibration.
• Detector parameters: These parameters affect the detector.
The following figure shows the location of the parameters on the ion optics path.

Figure 2-6 Ion Optics Path and Parameters

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 Principles of Operation

Location Parameter Parameter Use Scan Type

Type
1 IonSpray Source and gas The IS parameter controls the voltage All
Voltage (IS) applied to the electrode in the
®
TurboIonSpray probe that ionizes the
sample in the ion source. The parameter
depends on the polarity and it affects the
spray stability and the sensitivity. This
®
parameter is also used with the NanoSpray
ion source. Refer to the applicable ion
source Operator Guide. The parameter
can be compound-dependent and should be
optimized for each compound.
1 Nebulizer Source and gas The NC parameter controls the current All
Current (NC) applied to the corona discharge needle in
TM
the APCI probe, used in the Turbo V or
TM
DuoSpray ion source. The discharge
ionizes solvent molecules, which in turn
ionize the sample molecules.
1 Interface Source and gas The IHT parameter controls the temperature All
®
Heater of the NanoSpray interface heater and is
®
Temperature only available if the NanoSpray ion source
(IHT) and interface are installed.
The optimal heater temperature depends on
the type of sample being analyzed and the
solvent used. If the heater temperature is
too high, the signal degrades. Typically,
heater temperatures are in the 130 °C to
180 °C range. The maximum heater
temperature that can be set is 250 °C, but
this is too high for most applications.
1 Ion Source Source and gas The GS1 parameter controls the nebulizer All
®
Gas 1 (GS1) gas for both the TurboIonSpray and APCI
probes. The GS1 parameter controls the
®
nebulizer gas for the TurboIonSpray probe.
1 Ion Source Source and gas The GS2 parameter controls the heater gas All
®
Gas 2 (GS2) for the TurboIonSpray probe. The GS2
parameter controls the heater gas for the
®
TurboIonSpray probe and the nebulizer gas
for the APCI probe.

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Principles of Operation

Location Parameter Parameter Use Scan Type

Type
1 Temperature Source and gas The TEM parameter controls the All
(TEM) temperature of the heater gas for the
®
TurboIonSpray probe or the temperature
of the APCI probe.
1 Curtain Gas Source and gas The CUR parameter controls the gas flow of All
TM
(CUR) the Curtain Gas interface. The Curtain
TM
Gas interface is located between the
curtain plate and the orifice. It prevents the
contamination of the ion optics.
1 sdp Source and gas The sdp parameter controls the selection of n/a
TM
the DuoSpray ion source probes:
®
TurboIonSpray or APCI probe.
1 Declustering Compound The DP parameter controls the voltage on All
Potential the orifice, which controls the ability to
(DP) decluster ions between the orifice and the
®
QJet ion guide. It is used to minimize the
solvent clusters that might remain on the
sample ions after they enter the vacuum
chamber, and, if required, to fragment ions.
The higher the voltage, the higher the
energy imparted to the ions. If the DP
parameter is too high, then unwanted
fragmentation might occur.
Use the preset value and optimize for the
compound.
2 Entrance Compound The EP parameter controls the potential All
Potential (EP) difference between the voltage on Q0 and
ground. The entrance potential guides and
focuses the ions through the high-pressure
on the Q0 region.
Use the preset value.

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 Principles of Operation

Location Parameter Parameter Use Scan Type

Type
2 Q0 Trapping Compound The Q0 trapping parameter controls the EMS, EMC, EPI, ER,
storage of ions in the Q0 region. It is used and MS/MS/MS
to increase sensitivity and duty cycle by
trapping ions in the Q0 region while ions
are being mass-selectively ejected from the
linear ion trap. Use a fixed fill time with this
parameter.
Either select or clear the feature as required
by the experiment.
We recommend using a fixed fill of 20 ms
or greater.
3 CAD Gas Source and gas The CAD parameter controls the pressure of Q3 MI, Q3 MS, MRM,
the CAD gas in the collision cell during Q3, Prec, NL, EMS, ER,
MS/MS, and LIT scans. For Q3 scans, the EPI, MS/MS/MS,
collision gas helps to focus the ions as they EMC, and TDF
pass through the Q2 collision cell. The preset
value for the CAD parameter is in fixed
mode. For MS/MS scan types, the CAD gas
helps to fragment the precursor ions. When
the precursor ions collide with the collision
gas, they dissociate to form product ions.
For LIT scan types, the collision gas helps to
focus and trap ions in the linear ion trap.
Use the preset value and optimize for the
compound.
3 Collision Compound The CE parameter controls the potential EPI, MS/MS/MS,
Energy (CE) difference between the Q0 region and the MRM, MS2, Prec, NL,
Q2 collision cell. It is used only in MS/MS and LIT
scan types. This parameter is the amount of
energy that the precursor ions receive as
they are accelerated into the Q2 collision
cell, where they collide with gas molecules
and fragment.
Use the preset value and optimize for the
compound.

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Principles of Operation

Location Parameter Parameter Use Scan Type

Type
3 Collision Compound The CES parameter, in conjunction with the EPI and MS/MS/MS
Energy CE parameter, determines which three
Spread (CES) discreet collision energies are applied to the
precursor mass in an Enhanced Product Ion
(EPI) or MS/MS/MS (MS3) scan when CES
is used. When a collision energy spread
value is entered, CES is automatically turned
on.
Use the preset value and optimize for the
compound.
3 Collision Cell Compound The CXP parameter is only used in Q3 and Q3, MRM, MS2, Prec,
Exit Potential MS/MS scan types. This parameter transmits NL
(CXP) the ions into the Q3 quadrupole.
Use the preset value and optimize for the
compound.
4 Q3 Entry Compound The Q3 Entry Barrier parameter is used to EMS, EMC, EPI, ER,
Barrier transfer the ions from the Q2 collision cell and MS/MS/MS
into the linear ion trap.
Use the preset value.
4 Q3 Empty Compound The Q3 Empty Time parameter controls the EMC
Time amount of time that elapses before
singly-charged ions are removed from the
linear ion trap.
Use the preset value.
5 Multi-Charge Compound The MCS Barrier parameter controls the EMC
Separation voltage used to eliminate the singly-charged
(MCS) Barrier ions from the linear ion trap.
Use the preset value.
6 MS/MS/MS Compound The MS/MS/MS Fragmentation Time MS/MS/MS
Fragmentation parameter controls the amount of time that
Excitation the excitation energy is applied. It is used
Time in combination with the excitation energy
to fragment the isolated second precursor
ion.
Use the preset value.

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 Principles of Operation

Location Parameter Parameter Use Scan Type

Type
6 Fixed LIT Fill Compound The Fixed LIT Fill Time parameter controls EMS, EPI, ER, and
Time the amount of time that the LIT fills with MS/MS/MS
ions.
Use the preset value and adjust it to achieve
the desired signal response based on sample
concentration.
6 Dynamic Fill Compound The DFT parameter dynamically calculates EMS, EPI, ER, and
Time (DFT) the amount of time that ions are collected MS/MS/MS
in the linear ion trap based on the incoming
ion signal. When DFT is turned on, the signal
is optimized to either increase sensitivity or
minimize space-charging.
Either select or clear the feature based on
the experiment.
In the Tools > Settings > Method
Options dialog, the DFT settings are
optimized for the default scan rate. These
settings are also suitable for other LIT scan
speeds.
7 CEM Detector The CEM parameter controls the voltage All
applied to the detector. The voltage controls
the detector response.

®
Theory of Operation—Analyst Software
Quantitation
The goal of quantitation in LC-MS/MS is to accurately determine the concentration of a compound in an unknown
®
sample. The MRM scan type is primarily used for quantitative analysis in a triple quadrupole or QTRAP mass
spectrometer. In an MRM scan, the ability to define the precursor ion and characteristic product ion enables the
creation a pair that is highly specific of the analyte. The MRM transition (pair) coupled with the retention time
associated with the analyte during liquid chromatography provides the specificity required for quantitation.
Quantitation is accomplished through the use of validated MRM LC-MS/MS acquisition methods, acquisition of
calibration standard curves, and the subsequent integration of the peaks associated with the compounds of interest.
The calibration curve relationship between signal response and concentration is used to determine the quantity
of a particular analyte in an unknown sample.

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Principles of Operation

Integration
In LC-MS/MS data, integration refers to obtaining the area under a curve for the peak associated with a specific
compound. Through the development of a method which specifies the transitions, expected retention times,
internal standards, integration, and regression parameters, the software is able to automatically integrate peaks
for a given set of samples.

About Results Tables

Results tables summarize the calculated concentration of an analyte in each unknown sample based on the
calibration curve. Results tables also include the calibration curves as well as statistics for the results. The user
can customize the results tables and view the results tables in layouts.
The data from a results tables can be exported to a txt file for use in other applications, such as Microsoft Excel.
The user can also export data in the table or just the data in the visible columns.

Calibration Curves
A calibration curve, also know as a standard concentration curve, is a method for determining the concentration
of a substance in an unknown sample by comparing the unknown sample to a set of standard samples of known
concentration. The calibration curve is a plot of how the instrument responds (the analytical signal) to changes in
the concentration of the analyte (the substance to be measured). The operator prepares a series of standards
across a range of concentrations near the expected concentration of the analyte in the unknown sample.
Calibration standards are used to build calibration curves. Incorrect readings or missing readings on some of the
calibration samples might indicate issues with the analytical run. Follow acceptable methods found in literature
and regulatory agency guidances to create a calibration curve. Examples of good practice in the preparation of
calibration curves include:
• Preparing calibration standards in blank matrix in which the analyte is to be measured.
• Generating a calibration curve for each analyte to be measured.
• Making sure of the coverage of the expected concentration range of the analyte, including typical and atypical
specimens.
• Using six to eight standards to generate the curve.
This is not a comprehensive list and other guidances should be used in determining the best practice in developing
a calibration curve for the laboratory.

Note: In some analytical runs, single-point calibration standards are used. Single-point calibrations are performed
using a matrix blank sample and a single standard concentration. The relationship between instrument response
and analyte concentration is determined by the line created by these two points. Both the acquisition and
quantitation methods should be validated before being accepted for their intended use.

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 Principles of Operation

Regression
The area of the analyte peaks in the calibration curve standards is plotted against the known concentrations.
Subsequently, a line is fitted to the points. This regression line is used to calculate the concentration of the unknown
samples.
• Linear (y = mx + b)
• Linear through Zero (y = mx)
• Quadratic (y = a2 + bx + c)
As well, it is possible to add different types of weighting for the regression, including:
• 1/x
2
• 1/x
• 1/y
2
• 1/y

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Operating Instructions — Sample
Workflows 3
Table 3-1 Instrument Setup Workflow

Step To do this... Find the information in.... What does it do?

1 Create a hardware profile. Create a Hardware Profile on page 55 Each hardware profile must
include a mass
spectrometer and other
devices, such as an LC
system. Only devices
included in the active
hardware profile can be
used when creating
acquisition methods.
2 Create projects to store data. Create Projects and Subprojects on Using projects and
page 62 subprojects facilitates data
management and makes
comparison of results
easier.
3 Optimize the mass Verify Instrument Performance on page This is the process of
spectrometer. 66 optimizing the resolution
and mass spectrometer
parameters, and calibrating
the mass spectrometer to
obtain the best sensitivity
and performance from the
system.

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 Operating Instructions — Sample Workflows

Table 3-2 Sample Acquisition Workflow

Step To do this... Find the information in.... What does it do?

1 Create projects to store data. Create Projects and Subprojects on Using projects and
page 62 subprojects facilitates data
management and makes
comparison of results
easier.
2 Create an acquisition method. Operating Instructions — Acquisition To analyze samples, create
Methods on page 84 an acquisition method for
the mass spectrometer and
any LC devices. An
acquisition method
indicates which peripheral
devices to use, when to use
them to acquire data, and
the associated parameters.
3 Create and submit a batch. Add Sets and Samples to a Batch on After creating an
page 136 and Submit a Sample or Set acquisition method, run
of Samples on page 140 samples by creating an
acquisition batch and
submitting the batch to the
acquisition queue.
4 Run samples to acquire data. Acquire Data on page 140 Running samples involves
managing the acquisition
queue and monitoring
instrument and device
status. To submit samples
and acquire data, use the
Queue Manager. The Queue
Manager shows queue,
batch, and sample status,
and facilitates management
of samples and batches in
the queue.

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Operating Instructions — Sample Workflows

Table 3-2 Sample Acquisition Workflow (continued)

Step To do this... Find the information in.... What does it do?

5 Analyze data in Explore mode. Operating Instructions — Analyze and In Explore mode, many
Explore Data on page 110 tools are available for
—OR—
viewing and processing the
Step 6—Analyze quantitative acquired data. Graphs can
data. be customized with peak
labels and captions, contour
plots can be shown, and
spectra can be saved in the
library.
6 Analyze quantitative data. Operating Instructions — Analyze and Use the various quantitative
Process Quantitative Data on page 90 method creation tools in
Quantitate mode to analyze
the acquired data and build
a quantitative method to
generate a Results Table.
Use the Results Table to
manually review all of the
peaks for each analyte and
internal standard within a
batch and to view
calibration curves, sample
statistics, and metric plots.

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 Operating Instructions — Sample Workflows

Table 3-3 Example Routine Analysis Workflow

Step To do this... Find the information in.... What does it do?

1 Activate a hardware profile Create a Hardware Profile on page 55 Each hardware profile must
applicable to the method. include a mass
spectrometer and other
devices, such as an LC
system. Only devices
included in the active
hardware profile can be
used when creating
acquisition methods.
2 Create projects to store data. Create Projects and Subprojects on Using projects and
page 62 subprojects facilitates data
management and makes
comparison of results
easier.
3 Create and submit a batch. Add Sets and Samples to a Batch on After creating an
page 136 and Submit a Sample or Set acquisition method, run
of Samples on page 140 samples by creating an
acquisition batch and
submitting the batch to the
acquisition queue.
4 Equilibrate the system. Equilibrate the System on page 139 Equilibrate the system prior
to starting data acquisition.
A system that is not
equilibrated can result in
poor data.
5 Run samples to acquire data. Acquire Data on page 140 Running samples involves
managing the acquisition
queue and monitoring
instrument and device
status. To submit samples
and acquire data, use the
Queue Manager. The
Queue Manager shows
queue, batch, and sample
status, and facilitates
management of samples
and batches in the queue.

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Operating Instructions — Sample Workflows

Table 3-3 Example Routine Analysis Workflow (continued)

Step To do this... Find the information in.... What does it do?

6 Analyze data in Explore mode Operating Instructions — Analyze and In Explore mode, many
(optional). Explore Data on page 110 tools are available for
viewing and processing the
acquired data. Graphs can
be customized with peak
labels and captions,
contour plots can be
shown, and spectra can be
saved in the library.
TM TM
• For quantitative analysis using the MultiQuant software follow steps 7 to 8. The MultiQuant software is
recommended for larger data sets.
®
• For quantitative analysis using the Analyst software follow steps 9 to 10a
®
• For qualitative analysis using the Analyst software follow steps 9 to 10b.
TM
7 Analyze quantitative data in the MultiQuant Software Generate and use a Results
TM
MultiQuant software. Reference Guide: Chapter 7, 8, 10, Table to manually review
11, 12, 13, 14 all of the peaks for each
analyte and internal
standard within a batch
and to view calibration
curves, sample statistics,
and metric plots.
TM
8 Create a report in the MultiQuant Software Generate a report using the
TM
MultiQuant software. Reference Guide: Appendix C provided report templates
for the generated and
reviewed results.
9 Analyze qualitative (or Operating Instructions — Analyze and Generate a Results Table to
quantitative) data in the Process Quantitative Data on page 90 manually review all of the
®
Analyst software. peaks for each analyte and
internal standard within a
batch. For quantitative
analysis, also review
calibration curves, sample
statistics, and metric plots.

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 Operating Instructions — Sample Workflows

Table 3-3 Example Routine Analysis Workflow (continued)

Step To do this... Find the information in.... What does it do?

10a Create a report in the Analyst Generate Reports Generate a report using the
Reporter. provided report templates
for the generated and
reviewed results. For
reports specific to
qualitative analysis, use the
set of report templates
labeled with library
searching.
10b Select a library and then create Generate Reports Select the appropriate MS/
a report using the Analyst MS spectra library for the
Reporter. results and generate a
report using the provided
report templates labeled as
library search for the
generated and reviewed
results. For reports specific
to qualitative analysis, use
the set of report templates
labeled with library search.

Table 3-4 Example Method Developer Workflow

Step To do this... Find the information in.... What does it do?

1 Create a hardware profile. Create a Hardware Profile on page 55 Each hardware profile must
include a mass
spectrometer and other
devices, such as an LC
system. Only devices
included in the active
hardware profile can be
used when creating
acquisition methods.
2 Create projects to store data. Create Projects and Subprojects on Using projects and
page 62 subprojects facilitates data
management and makes
comparison of results
easier.

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Table 3-4 Example Method Developer Workflow (continued)

Step To do this... Find the information in.... What does it do?

3 Optimize the compound Automatic Optimization on page 69 The software automatically
automatically optimizes the compound
and mass spectrometer
- Or - Step 4 Optimize the
parameters for the
compound manually.
compounds of interest.
4 Optimize the compound Operating Instructions — Manual The user manually
manually. Compound Optimization on page 164 optimizes the compound
and mass spectrometer
parameters for the
compounds of interest.
Manual optimization
provides more experienced
users with greater control
during the optimization
process.
5 Create an acquisition method. Operating Instructions — Acquisition To analyze samples, create
Methods on page 84 an acquisition method for
the mass spectrometer and
any LC devices. An
acquisition method
indicates which peripheral
devices to use, when to use
them to acquire data, and
the associated parameters.
6 Create and submit a batch. Add Sets and Samples to a Batch on After creating an
page 136 and Submit a Sample or Set acquisition method, run
of Samples on page 140 samples by creating an
acquisition batch and
submitting the batch to the
acquisition queue.
7 Equilibrate the system. Equilibrate the System on page 139 Equilibrate the system prior
to starting data acquisition.
A system that is not
equilibrated can result in
poor data.

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Table 3-4 Example Method Developer Workflow (continued)

Step To do this... Find the information in.... What does it do?

8 Run samples to acquire data. Acquire Data on page 140 Running samples involves
managing the acquisition
queue and monitoring
instrument and device
status. To submit samples
and acquire data, use the
Queue Manager. The
Queue Manager shows
queue, batch, and sample
status, and facilitates
management of samples
and batches in the queue.
9 Analyze data in Explore mode Operating Instructions — Analyze and In Explore mode, many
(optional). Explore Data on page 110 tools are available for
viewing and processing the
acquired data. Graphs can
be customized with peak
labels and captions,
contour plots can be
shown, and spectra can be
saved in the library.
TM TM
• For quantitative analysis using the MultiQuant software follow steps 10 to 12. The MultiQuant software
is recommended for larger data sets.
®
• For quantitative analysis using the Analyst software follow steps 13 to 15.
• For qualitative analysis, contact Support.
TM
10 Create a quantitative method MultiQuant Software Use the various
TM
in the MultiQuant software. Reference Guide: Quantitation quantitative method
Method Editor creation tools in the
software to analyze the
acquired data and build a
quantitative method to
generate a Results Table.

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Operating Instructions — Sample Workflows

Table 3-4 Example Method Developer Workflow (continued)

Step To do this... Find the information in.... What does it do?

TM
11 Analyze quantitative data in MultiQuant Software Generate and use a Results
TM
MultiQuant software. Reference Guide: Chapter 7, 8, 10, Table to manually review
11, 12, 13, 14 all of the peaks for each
analyte and internal
standard within a batch
and to view calibration
curves, sample statistics,
and metric plots.
TM TM
12 Create a report in MultiQuant MultiQuant Software Generate a report using the
software. Reference Guide: Appendix C provided report templates
for the generated and
reviewed results.
TM
13 Create a quantitative method MultiQuant Software Use the various
®
in the Analyst software. Reference Guide: Quantitation quantitative method
Method Editor creation tools in the
software to analyze the
acquired data and build a
quantitative method to
generate a Results Table.
14 Analyze qualitative (or Operating Instructions — Analyze and Generate a Results Table to
®
quantitative) data in Analyst Process Quantitative Data on page 90 manually review all of the
software. peaks for each analyte and
internal standard within a
batch. For quantitative
analysis, also review
calibration curves, sample
statistics, and metric plots.
14 Create a report in the Analyst Generate Reports Generate a report using the
Reporter. provided report templates
for the generated and
reviewed results. For
reports specific to
qualitative analysis, use the
set of report templates
labeled with library
searching.

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Operating Instructions—Hardware 4
WARNING! Personal Injury Hazard. Follow the instructions in the documentation
when using the system. The protection provided by the equipment might be impaired
if the equipment is used in a manner not specified by SCIEX.

Start Up the System

WARNING! Electrical Shock Hazard. Make sure that the system can be
disconnected from the mains supply outlet in an emergency. Do not block
the mains supply outlet.

WARNING! Lifting Hazard. Do not move the system. Risk of personal injury
or system damage. If the system must be moved, then contact a Field Service
Employee (FSE).

Note: Before operating the instrument, read the safety information in Operational Precautions and Limitations
on page 8.

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Operating Instructions—Hardware

Prerequisites
• The site requirements specified in the Site Planning Guide are met. The Site Planning Guide includes
information on the mains supply and connections, compressed air, nitrogen, roughing pump, ventilation,
exhaust, and site clearance requirements. Contact us for a copy of the Site Planning Guide, if required.
For contact information, go to sciex.com/contact-us.
• The source exhaust gas, compressed air, and nitrogen gases are connected to the mass spectrometer.
• The 4 L source exhaust drain bottle is connected to the exhaust waste connection on the back of the mass
spectrometer and to the laboratory ventilation system.
• The source exhaust hoses are securely clamped at the mass spectrometer, drain bottle, and ventilation
connections.
• The mass spectrometer convenience switch is turned off and the mains supply cable is plugged into the mass
spectrometer.
• The mass spectrometer and roughing pump mains supply cables are plugged into the 200 VAC to 240 VAC
mains supply.
• The Ethernet cable is connected to both the mass spectrometer and the computer.

1. Turn on the mass spectrometer convenience switch. Refer to Figure 2-2 on page 23.
2. Turn on the computer.
®
3. Open the Analyst software.

Reset the Mass Spectrometer

1. Stop any ongoing scans and then turn off sample flow to the mass spectrometer.
®
2. In the Analyst software, deactivate the hardware profile, if it is active.
3. Press and hold the Reset button for five seconds.

A click is heard when the relay activates. After approximately three minutes, the mass spectrometer reaches
operating pressure.

Shut Down and Vent the System

Some procedures require that the system be shut down. Others require that it also be vented. Follow these steps
to shut down and, if required, vent the system.

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 Operating Instructions—Hardware

Note: If the input gas supply must be disconnected, then relieve the pressure in the gas lines before disconnecting
it.

Tip! If the mass spectrometer will not be used for a length of time, then leave it in Standby mode with the ion
source in place. If the mass spectrometer must be shut down, then follow these instructions. Do not turn off the
roughing pump until after the turbo pumps have spun down.

1. Complete or stop any ongoing scans.

CAUTION: Potential System Damage. Turn off the sample flow before shutting down
the system.

2. Turn off the sample flow to the system.

®
3. In the Analyst software, deactivate the hardware profile, if it is active.
4. Close the software.
5. (If required) Follow these steps to vent the system:

Note: Vent the system before performing a full cleaning of the vacuum interface, before cleaning the Q0
region, and before replacing the roughing pump oil. For more information contact the Qualified Maintenance
Person (QMP) or FSE.

a. Press and hold the Vent button for three seconds.

The Vacuum LED begins flashing quickly (more quickly than during pump down). The turbo pump spins
down gradually.
b. Allow the system to vent for 15 minutes.
6. Turn off the mass spectrometer convenience switch.
7. Disconnect the mass spectrometer mains supply cable from the mains supply outlet.
8. (If venting the system) Disconnect the roughing pump mains supply cable from the mains supply outlet.

Adjust the Integrated Syringe Pump Position

WARNING! Puncture Hazard. Take care when handling the syringe. The tip
of the syringe is extremely sharp.

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Operating Instructions—Hardware

WARNING! Puncture Hazard. Make sure that the syringe is seated properly
in the syringe pump and that the automatic syringe pump stop is adjusted
properly to avoid damaging or breaking the glass syringe. If the syringe
breaks, follow established safety procedures for sharps disposal.

1. Press the Release button on the right side of the syringe pump to lower the base and then insert the syringe.
Refer to Figure 4-1.

Figure 4-1 Lowering the Syringe

Item Description
1 Syringe plunger
2 Release button. Press to raise or lower the base.

2. Make sure that the end of the syringe is flush against the base and that the shaft of the syringe rests in the
cutout.

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 Operating Instructions—Hardware

3. Adjust the post so that it triggers the automatic syringe stop before the syringe plunger hits the bottom of the
glass syringe. Refer to Figure 4-2.

Figure 4-2 Automatic Syringe Stop

Item Description
1 Automatic syringe stop. After the post hits the automatic syringe stop,
the syringe pump stops.
2 Post. Adjust the height to prevent the syringe plunger from hitting the
syringe during sample infusion.
3 Post lock screw. Tighten the screw after the height of the post is adjusted.

4. Turn the side screws as shown in Figure 4-3 to secure the syringe.

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Operating Instructions—Hardware

Figure 4-3 Syringe Pump Screws

5. Make sure that the mass spectrometer and integrated syringe pump are activated in the software.

Note: For subsequent manual use, on the mass spectrometer, press the button on the right side of the
syringe pump to start the flow. Refer to Figure 4-4. The LED beside the button flashes when the syringe pump
®
is in use. The syringe pump flow can also be controlled automatically by the Analyst software.

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 Operating Instructions—Hardware

Figure 4-4 Syringe Pump LED

Item Description
1 Syringe pump on and off button
2 Syringe pump status LED
®
6. In the Analyst software, on the Navigation bar, double-click Manual Tuning.
7. Click Start Syringe.
8. To stop the syringe pump, click Stop Syringe.

Plumb the Diverter Valve

The integrated diverter valve, which is located next to the ion source, can be plumbed in injector mode or diverter
mode. To configure the valve, access the Configuration tab and then make sure that the Use integrated
injector/diverter valve check box is selected. Refer to Add Devices to a Hardware Profile on page 59.

CAUTION: Potential Wrong Result. Do not press the diverter valve button during a run.
Doing so might result in incorrect data.

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Operating Instructions—Hardware

Plumb the Diverter Valve in Injector Mode

When the valve is in Position A, the sample flows through the external loop. When the valve switches to Position
B, the sample is injected.
• Plumb the valve for injector mode.

Figure 4-5 Diverter Valve—Injector Mode Position A

Figure 4-6 Diverter Valve—Injector Mode Position B

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 Operating Instructions—Hardware

Item Description
1 Sample in
2 Waste out
3 Sample loop (ports 3 and 6)
4 Mobile phase in
5 To column (or to the mass spectrometer, if a column is not installed)

Plumb the Diverter Valve in Diverter Mode

When the valve is in Position A, the flow goes to waste. When the valve switches to Position B, the flow goes to
the mass spectrometer.
• Plumb the valve for diverter mode.

Figure 4-7 Diverter Valve—Diverter Mode Position A

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Figure 4-8 Diverter Valve—Diverter Mode Position B

Item Description
1 To mass spectrometer
2 From column
3 Waste out

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Operating Instructions —
Hardware Profiles and Projects 5

Hardware Profiles
A hardware profile tells the software how the mass spectrometer and the devices are configured and connected
to the computer. Multiple hardware profiles can be set up, but only one profile can be active at any time.
When a hardware profile is created in the Hardware Configuration Editor, the peripheral devices must be configured
so that the software can communicate with them. Configuring the peripheral devices requires two procedures:
setting up the physical connections and configuring the software to communicate with the peripheral devices.
When the software is installed, the driver required for each peripheral device is also installed. After the peripheral
devices are physically connected to the computer, set up the appropriate configuration information.
Each hardware profile must include a mass spectrometer. Before creating an acquisition method, make sure that
all devices to be used in the method are included in the hardware profile, including the syringe pump. The devices
configured in the active hardware profile and selected in the Add/Remove Device Method dialog are shown as
icons in the Acquisition method pane. Only peripheral devices included in the active hardware profile can be used
when creating acquisition methods.
Each hardware profile must include a mass spectrometer. Before creating an acquisition method, make sure that
all devices to be used in the method are included in the hardware profile. The devices configured in the active
hardware profile and selected in the Add/Remove Device Method dialog are shown as icons in the Acquisition
method pane. Only peripheral devices included in the active hardware profile can be used when creating acquisition
methods.
For information about setting up the physical connections to the devices, refer to the Peripheral Devices Setup
®
Guide. For a list of the supported devices, refer to the Software Installation Guide for the Analyst software.

Create a Hardware Profile

The user can create multiple hardware profiles, but only one profile can be active at any time.

1. On the Navigation bar, under Configure, double-click Hardware Configuration.

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Operating Instructions — Hardware Profiles and Projects

Figure 5-1 Hardware Configuration Editor Dialog

2. In the Hardware Configuration Editor dialog, click New Profile.

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 Operating Instructions — Hardware Profiles and Projects

Figure 5-2 Create New Hardware Profile Dialog

3. Type a name in the Profile Name field.

4. Click Add Device.

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Operating Instructions — Hardware Profiles and Projects

Figure 5-3 Available Devices Dialog

In the Available Devices dialog, in the Device Type field, Mass Spectrometer is the preset value.
5. In the Devices list, select the appropriate mass spectrometer and then click OK to return to the Create New
Hardware Profile dialog.
6. Click Setup Device.
7. (Optional) To configure mass spectrometers that use the integrated syringe pump, on the Configuration
tab, select the Use integrated syringe pump check box.
8. (Optional) To configure the mass spectrometer for the diverter valve, on the Configuration tab, select Use
integrated injector/diverter valve.
9. (Optional) Select additional features on the Configuration and Communication tabs as required.
10. Click OK to return to the Create New Hardware Profile dialog.
11. Click Add Device and then add and set up each device that is used with the mass spectrometer. Refer to
Add Devices to a Hardware Profile on page 59.
12. Click OK in the Create New Hardware Profile dialog.
13. Click the hardware profile to be activated in the Hardware Configuration Editor.
14. Click Activate Profile.

The check mark turns green. If a red x is shown, then there is an issue with the hardware profile activation.

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Tip! A hardware profile need not be deactivated before another is activated. Click a hardware profile and
then click Activate Profile. The other profile is deactivated automatically.

15. Click Close.

Add Devices to a Hardware Profile

Devices must be configured to enable the software to communicate with them. When the software is installed,
the driver required for each device is also installed. After the devices are physically connected to the computer,
configure them.
Only the devices configured in the active hardware profile and selected in the Add/Remove Device Method dialog
are shown as icons in the Acquisition Method Browser pane.
1. Open the Hardware Configuration Editor.
2. In the Hardware Profiles list, deactivate the hardware profile.
3. Click Edit Profile.
4. Click Add Device.

Note: Remember to add a mass spectrometer. For more information, refer to Create a Hardware Profile on
page 55.

5. In the Available Devices dialog, in the Device Type list, select the device and then click OK.

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Operating Instructions — Hardware Profiles and Projects

Figure 5-4 Available Devices Dialog

6. Click OK.
7. Select the device from the Devices list and then click OK.
8. Click Setup Device.

A dialog containing configuration values for the device opens.

9. (Optional) On the Communication tab, in the Alias field, type a name or other identifier for the device.

Note: For devices using serial communication, make sure that the serial port selected matches the serial
port to which the device is physically connected. When the serial expansion cable is used, the number selected
in the profile is the number on the cable plus two.

Note: The Alias field might also be referred to as the Name box and might be found on another tab under
Alias.

• If the device uses a Serial Port as a communication interface, then in the COM Port Number list,
select the COM port to which the device is connected.

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• If the device uses Ethernet as a communication interface, then type the IP Address assigned to the
device by the administrator or use the corresponding Host Name for the address.
• If the device uses GPIB Board as a communication interface, then do not change the settings for the GPIB
board.
The rest of the preset values for the device are probably appropriate. Do not change them. For information
about the Configuration and Communication tabs, refer to the Help.
10. To restore the device preset values, on the Communication tab, click Set Defaults.
11. To save the configuration, click OK.
12. Repeat step 4 to step 11 for each device.
13. Click OK in the Create New Hardware Profile dialog.
14. To activate the hardware profile, in the Hardware Configuration Editor, click the hardware profile.
15. Click Activate Profile.

The check mark turns green. If a red x is shown, then there is an issue with the hardware profile activation.
For more information, refer to Troubleshoot Hardware Profile Activation on page 61.

Tip! An active hardware profile does not have to be deactivated before another one is activated. Click an
inactive hardware profile and then click Activate Profile. The other profile is deactivated automatically.

16. Click Close.

Troubleshoot Hardware Profile Activation

If a hardware profile fails to become active, then a dialog opens indicating which device in the profile failed. A
device might fail to activate because of communications errors.
1. Read the error message generated. Depending on the message, there might be an issue with a device or how
the communication is set up.
2. Verify that the device has power and is turned on.
3. Verify that the COM port or IP address assigned to the device is correct.

Tip! On computers with two built-in serial ports, the first port on the serial port expansion card is usually
COM3, even though the cable indicates P1.

4. Verify that the communication settings for the device (for example, dip switch settings) are set correctly and
match the settings on the Communication tab.
5. Turn off the device.
6. Wait 10 seconds.

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Operating Instructions — Hardware Profiles and Projects

7. Turn on the device.

Wait until all device power-up activities are complete before trying to activate the hardware profile again.
Some devices might require 30 seconds or more to complete the power-up activities.
8. Activate the hardware profile.
9. If the issue persists, then delete the failing profile and create a new one.
10. If the issue still persists, then contact technical support.

Projects and Subprojects

Before beginning an experiment, decide where to store the files related to the experiment. Use projects and
subprojects for each experiment to better manage data and compare results. For example, use subprojects to store
the results for specific dates.

Create Projects and Subprojects

To use a subproject structure within a project, create the subproject structure when the project is created.

1. Click Tools > Project > Create Project.

Figure 5-5 Create New Project/Subproject Dialog

Note: A new subproject cannot be created for a project that was not originally created with a subproject.

2. Type a project name in the Project name field.

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3. (Optional) To use subprojects, select the required folders and then use the arrow buttons to move them to the
Subproject folders list.
4. (If subprojects are used) In the Subproject name field, type a name for the first subproject or use the existing
date.
5. (Optional) To use this project and subproject folder organization for all new projects, select the Set
configuration as default for new projects check box.

All new projects are created with this folder configuration.

6. Click OK.

Create Subprojects
Subprojects can only be created in a project that has an existing subproject structure.

1. On the Project tool bar, from the Project list, select the project.
2. Click Tools > Project > Create Subproject.
3. In the Subproject name box, type a name for the subproject or use the existing date.
4. Click OK.

Copy Subprojects
A subproject can be copied from another project that has existing subprojects. If the copied subprojects contain
folders that also exist in the project folder, then the software uses the project level folders.
1. Click Tools > Project > Copy Subproject.

The Copy Subproject dialog is shown.

2. Click Browse to navigate to the subproject source.
3. Click OK.
4. Select the subproject from the Source Subproject list.
5. Click Browse to navigate to the subproject destination.
6. Type the name in the Target Subproject field.
7. Click OK.
8. Do one of the following:

• To copy all folders and files from the Subproject Source into the Subproject Destination, select
the Copy Contents check box.

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Operating Instructions — Hardware Profiles and Projects

• To copy only the folders in the same structure into the Subproject Destination, make sure that the
Copy Contents check box is cleared.

9. Click Copy.

Switch Between Projects and Subprojects

• On the software tool bar, from the project list, click the required project or subproject.

Figure 5-6 Project List

The project list in this figure shows the API Instrument, Default, and Example folders.

Installed Project Folders

Three project folders are installed with the software: API Instrument, Default, and Example.

API Instrument Folder

The API Instrument folder is unique and very important to the correct functioning of the mass spectrometer. The
API Instrument folder contains the information required for tuning and calibrating the mass spectrometer. This
information includes parameter settings files, reference files, instrument data files that contain calibration and
resolution information, and the acquisition methods used during automatic tuning. The API Instrument folder also
contains data files for manual tuning runs that were performed using the Start button rather than the Acquire
button. These data files are saved automatically in the API Instrument folder in the Tuning Cache folder and named
with the date and time they were created. The Tuning Cache folder is automatically cleared periodically.

Default Folder
The Default folder contains folders that are present in new projects and serves as a template for new projects.

Example Folder
The Example folder contains sample methods and data files. Users can practice working with the Explore or
Quantitate modes using the example data files. The example files are sorted into subfolders by mass spectrometer
type and application area.

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Back Up the API Instrument Folder

Back up the API Instrument folder regularly and after routine maintenance has been performed.
• Copy the API Instrument folder, paste it to a different location, preferably to another computer, and then
rename the folder. Use the date and a mass spectrometer reference if there is more than one mass spectrometer
when the folder is named. For example, API Instrument_instrument model3_010107

Recover the API Instrument Folder

Back up the API Instrument folder regularly and after routine maintenance has been performed. To recover
the API Instrument folder, do the following:
1. Rename the current API Instrument folder.
2. Copy the backup folder into the Projects folder.
3. Change the name of the backup folder to API Instrument.

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Tune and Calibrate 6
Run the Verify instrument performance option weekly or after the mass spectrometer is cleaned to confirm
that the system is working properly. In general, for triple quadrupole systems, calibration and resolution is
®
maintained for three to six months unless the system loses vacuum. For QTRAP systems, the resolution should
also be maintained for three to six months but the system should be calibrated approximately monthly. If the
system loses vacuum, then check the calibration and resolution before using the system. For more information
about tuning and calibration, refer to the Advanced User Guide and the Manual Tuning Tutorial.

Tip! Perform maintenance tasks regularly to make sure that the mass spectrometer is performing optimally.

Prerequisites
• The spray is stable and the correct tuning solution is being used.
• A printer is configured.

Required Materials
• Tuning solutions that are supplied in the Standards Chemical Kit shipped with the system. If required, a new
kit can be ordered from SCIEX.
• 5 mL, 1 mL, and 250 µL serial gas-tight syringes.
• Red PEEK sample tubing.

Verify Instrument Performance

The following procedure describes how to verify or adjust the performance of the mass spectrometer. For information
about using the other instrument performance options, refer to the Help. Refer to Calibration Ions and Solutions.
1. On the Navigation bar, under Tune and Calibrate, double-click Manual Tuning.
2. Start the syringe pump and then run a calibration method. Confirm that the Total Ion Chromatogram (TIC) is
stable and that the peaks of interest are present in the spectrum.
3. On the Navigation bar, under Tune and Calibrate, double-click Instrument Optimization.

The Instrument Optimization dialog opens.

4. Click Verify instrument performance.
5. Click Next.

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 Tune and Calibrate

6. Click Approved Tuning.

7. Click Next.
8. Select a Tuning Solution from the list.

Depending on the solution selected, different modes are available:

a. Click a polarity.
b. (If available) Click Q1 and Q3 in the Quad section.
c. (If available) Click the required scan speeds.
d. (If available) Click the scan speeds in the LIT section.
e. (If available) Click Excitation in the MS/MS/MS section
9. Click Next.
10. If the Select a mode page opens, then select Automatic.
11. Click Next.
12. Click GO.

The Verifying or Adjusting Performance dialog opens. After the process has completed, the Results Summary
opens. For more information, refer to the Help.
13. If applicable depending on the options selected. Change solutions for different scan types and polarities when
prompted.

About the Verifying or Adjusting Performance

Dialog
The top left corner shows the part of the instrument that is being tuned.
The Current Spectrum graph shows the spectrum of the current scan, the optimal scan selected by the software,
or the scan at the current parameter value when the software results are viewed in interactive mode.
The Instrument Optimization Decision Plots, in the top right graph, dynamically show the intensity versus voltage
curves of the parameters that are currently being optimized.

Results Summary
The Results Summary is a record of any instrument settings changes that were made by the Instrument Optimization
wizard.

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Tune and Calibrate

Figure 6-1 Results Summary

The Results Summary includes the location of data files and instrument settings backups, as well as step-by-step
changes and results during optimization.
The Results Summary also shows a verification report. This report contains a snapshot of the mass spectrum for
each relevant mass for the scan modes being verified. The spectrum is labeled with the target mass, where the
mass was found, mass shift, peak width, and peak intensity. The spectrum can be used as a visual record of peak
shape or scan mode performance. A summary table of results follows the spectra.
The Results Summary is automatically saved in the following path: <drive>:\Analyst Data\Projects\API
Instrument\Data\Instrument Optimization\yyyy-mm-dd\results.doc, where yyyy-mm-dd is the date on which the
report was created. Users can print the Results Summary or open a previously saved Results Summary.

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Automatic Optimization 7
Users will learn how to:
• Automatically optimize for the analyte using the Compound Optimization wizard.
• Choose between infusion and flow injection analysis (FIA).
• Optimize parameters:
• If infusion analysis is chosen, then use an infusion method to optimize compound-dependent parameters.
• If FIA analysis is chosen, then use FIA to optimize compound- and ion source-dependent parameters.
This tutorial uses minoxidil, tolbutamide, reserpine, and rescinnamine as example compounds. Other available
compounds can be used, but the methods must be adjusted accordingly.
The user can also optimize compounds manually.

Prerequisites
• The mass spectrometer is tuned and calibrated.
• (For FIA analysis) An acquisition method template is available.
• (For FIA analysis) An LC pump and an autosampler are connected and configured in the hardware profile.
• If the system has an integrated syringe pump, a syringe pump is configured in the hardware profile.
• All of the required peripheral devices, including the LC components, if needed, are configured in the hardware
profile.

Required Materials
• A syringe, preferably a 1.0 mL syringe.
• (For FIA analysis) Mobile phase: 1:1 acetonitrile:water + 2 mM ammonium acetate + 0.1% formic acid.

Note: Users can choose a different mobile phase based on the experimental properties of the compound.

• LC pump and autosampler.

• (For FIA Analysis) Autosampler vials.

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Automatic Optimization

Table 7-1 Compounds and Molecular Weights

Compound m/z
Minoxidil 210.2
Tolbutamide 271.3
Reserpine 609.4
Rescinnamine 635.4

Table 7-2 Estimates for Starting Concentrations

System Concentration
®
QTRAP 5500 system 10 ng/mL
TM
SCIEX Triple Quad 5500 system 10 ng/mL

About Automatic Optimization

Automatic optimization first checks for the presence of the compounds. The voltages of the various ion path
parameters are gradually increased or decreased to determine the maximum signal intensity (Q1 scan) for each
ion. A text file is generated and shown during the optimization process. This file records the various experiments
performed and the optimal values for each parameter. A file folder containing all the experiments performed is
also generated and can be found by opening the data file folder in Explore mode. For each experiment performed,
an acquisition method is also generated and saved in the Acquisition Method folder.
During the optimization process, select how the precursor ion and the corresponding product ions are to be chosen.

Types of Sample Introduction

Infusion
Infusion is the continuous flow of the sample at low flow rates into the ion source using a syringe pump. During
the infusion optimization process, the software can select precursor and product ions and optimize for declustering
potential, collision energy, and collision cell exit potential. The voltages of the ion path parameters are gradually
increased or decreased to determine the maximum signal intensity for the precursor and product ions.
Use infusion optimization to optimize compound-dependent parameters only at much lower flow rates than those
used during LC-MS/MS analysis.

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FIA
FIA is the injection of a sample by the autosampler into the mass spectrometer using LC. During the FIA optimization
process, multiple sample injections are performed for various ion source- or compound-dependent parameter types
that are changed between injections. FIA compound optimization optimizes parameters by performing looped
experiments in succession. One compound-dependent parameter is optimized first, followed by the next
compound-dependent parameter. FIA optimizes for ion source-dependent parameters by making one injection for
each value.
Compound parameters must be narrowed down using at least two more FIA cycles. Use FIA optimization to
optimize both compound-dependent and source-dependent parameters using LC at higher flow rates.

Table 7-3 Differences Between Sample Introduction Methods

Method Required Devices Parameters Typical Flow Rate

Range
Infusion Syringe pump Compound-dependent 5 µL/min to 25 µL/min
FIA LC pump and autosampler Source- and compound-dependent 25 µL/min to 1000 µL/min

During optimization, a text file is generated and then shown. This file records the various experiments performed
and the optimal values for each parameter. A file folder containing all the data files is also generated. For each
experiment performed, an acquisition method is also generated and saved in the Acquisition Methods folder.

Automatically Optimize for an Analyte Using

Infusion
In this section, users will perform automatic MS/MS optimization using infusion with a known precursor ion and
an unknown product ion.

Confirm the Presence of Compounds

Confirm the presence of compounds of interest before continuing with automatic optimization.
®
1. In the Analyst software, create a project.
2. Activate the hardware profile.
3. Infuse the compound in solution at a rate of 5 µL/min. to 10 µL/min.
4. On the Navigation bar, under Tune and Calibrate, double-click Manual Tuning.
5. In the method list field, click Syringe Pump Method.
6. On the Syringe Pump Method Properties tab, type the appropriate parameter values. Refer to Table 7-4.

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Automatic Optimization

Table 7-4 Syringe Pump Method Properties Tab

Parameter Typical Value

Syringe Diameter Syringe dependent; 1.0 mL syringe is 4.610 mm
Flow Rate 10
Unit µL/min.

Figure 7-1 Syringe Pump Method Properties Tab

7. Click Start Syringe Pump.

8. Click MS Method from the method list.
9. Click Start.
10. Wait until an even TIC is shown on the left and peaks are shown on the right and then click Stop.
11. Select the MCA check box.
12. Type 10 In the Cycles field.
13. Click Start.

When the ten scans are complete, the graph should show the masses of the four compounds as ions.

Note: The intensities of the compounds should be much higher than the smallest noise peaks but not so
high that any noise peaks are not seen. In the first case, the peak might not be a real compound. In the
second case, the concentration might be too high for the software to optimize properly.

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 Automatic Optimization

Figure 7-2 Compound Ions

Perform Automatic MS and MS/MS Optimization Using Infusion

with a Known Precursor Ion and an Unknown Product Ion
Automatic optimization for MS/MS analysis optimizes certain compound-dependent parameters for one or more
MRM transitions. The software finds the ion of interest and then optimizes the compound-dependent parameters
to get the maximum sensitivity for the compound. The software ramps CE and then selects the most intense
fragments that meet all of the product ion selection criteria.
If the initial Q1 scan signal is too high, then the software attempts to reduce the CEM to keep ions within the
detector range. If the signal is still too high after the CEM is reduced, then the process stops and an error message
is shown. Dilute the solution and then restart the optimization. Be sure to purge the infusion line. The parameters
from the last quantitative optimization are stored.
1. Make sure that the proper concentration of solution has been installed in the syringe pump, and that the syringe
pump has been started. If an integrated syringe pump is available and has been started, then the Syringe Pump
Status LED is flashing.

The syringe pump must be started in the Manual Tune window before compound optimization begins.
2. On the Navigation bar, under Tune and Calibrate, double-click Compound Optimization.
3. On the Instrument Settings page, in the Inlet section, click Infusion.
4. Click MS/MS Analysis in the Mass Spectrometer section.

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Automatic Optimization

5. Click Next.
6. On the Ions to use in MS/MS Analysis page, select the appropriate parameter values. Refer to Table 7-5.

Table 7-5 Example Parameters to use in MS/MS Analysis Page

Parameter Value
MW Ion: Search Window 2.500
Resolution Unit
Polarity Positive
Product Ion Auto Select
Resolution Unit

Note: The optimization algorithm looks for the most intense peak in the specified search window. If the
most intense peak in that window is not the mass of interest, then the software optimizes on the wrong ion.

7. Click Criteria next to the Auto Select option.

8. In the Product Ion Auto Selection Criteria dialog, type the appropriate parameters. Refer to Table 7-6.

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Table 7-6 Example Product Ion Auto Selection Criteria Dialog Parameters

Parameter Parameter Value

From the Most 3 The number of fragment peaks to be optimized. The algorithm
Intense (peaks) generates a product ion scan spectrum while ramping the CE in
MCA mode. In this example, the algorithm takes the three most
intense fragment ions from the spectrum and then continues the
MS/MS optimization on those fragments only.
For unknown compounds, select more peaks.
Build final method 2 The number of fragment ions per precursor ion (target compound)
using (most intense to be automatically included in the acquisition method. The
peaks) specified number defines the number of MRM transitions to be
included for each target compound in the method. The order of
preference is based on the intensity of the fragment ion.
Two is a better starting value than one because typically two
product ions are required for quantitation. Start with three in
case there is a issue with one of the two best. Go back and the
third is already identified.
For unknown compounds, select more peaks for use in case of
interference.
Exclude Product Ions 20.000 The Da value that defines the exclusion window around the
within ± (Da of precursor ion so that fragment ions that fall within this window
Precursor Ion m/z) are not selected for MRM optimization. For example, if the user
types ± 5 Da for a precursor ion that has an m/z of 500, then
any fragment ions within an m/z of 495 to 505 are excluded.
This prevents the precursor ion from being optimized as a product
ion.
Min. Mass for 60.000 The lowest fragment mass to be considered for optimization. Use
Product Ion (Da) this option to narrow or widen the window of fragment ions to
be considered from the precursor mass.
Threshold for 100.000 Minimum number of counts for a product ion to be considered.
Product Ion (cps)

9. Click OK to save the changes to the selection criteria.

10. Click Next.
11. In the Target Components dialog, type the appropriate parameter values. Refer to Table 7-7.

Note: The compound name must be unique for each compound or transition.

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Automatic Optimization

Table 7-7 Example Target Compounds Dialog Parameters

Target Field Value

Compound
Reserpine Compound Name Reserpine
MW (Da)* 609.3
No. Charges 1
Minoxidil Compound Name Minoxidil
MW (Da)* 210.2
No. Charges 1
Tolbutamide Compound Name Tolbutamide
MW (Da)* 271.3
No. Charges 1
Rescinnamine (IS) Compound Name Rescinnamine
MW (Da)* 635.4
No. Charges 1
*Type the exact ion mass.

12. Click Finish to begin the optimization process.

The screen shows two windows, a text file window and an acquisition window. The user might need to minimize
one of them to see the other. The experiment being run is shown on the top of the acquisition window. 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 Components
page.

Review the Optimization Results

At the end of the optimization, the optimized parameters are saved in an acquisition method. All dam and wiff
files generated in the optimization process are saved in the Acquisition Methods folder and in a subfolder in the
Data folder, respectively, in the project. The name of the subfolder is generated using the name of the compound
and the date.
1. After completing the optimization, print the text file containing the optimized parameters for each compound.
2. Click File > Open and then select the Reserpine_QOpt_FinalMRM.POS.dam file.

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3. Compare the values in the text file to those in the dam file.
4. Inspect the contents of the following folders:

• Data: Look through all of the runs executed during optimization. Compare a wiff file with the optimized
value in the method or printed parameters.
• Acquisition Method: Reserpine_QOpt_FinalMRM.POS.dam file and other dam files created during
optimization.
• Log: Report file (rtf) shown during the optimization process.

Automatically Optimize for an Analyte Using FIA

Prerequisites
• Identify the ions for the compounds and save the basic acquisition method.
• Add an autosampler and LC pump to the basic acquisition method. Using FIA for optimization requires that
these devices be active in the hardware profile.
• Create an LC-MS/MS acquisition method based on the Reserpine_QOpt_FinalMRM.POS.dam file and then
name the new method FIA_AutoOpt_Tutorial.dam.
• Make sure that the selected project contains the acquisition method that is being used.

Note: Although FIA can be used to optimize compound-dependent parameters, this is typically not done because
of the number of cycles required to obtain the optimal parameter values.

1. Put a dilution of the four-compound mix in an autosampler.

Enough sample is required to review each variable of each parameter and have sample left over. For example,
for the temperature to run at 300 °C, 400 °C, and 500 °C, if the injection volume is 10 µL, then more than a
30 µL (3 × 10 µL injection) is required.
2. Confirm that LC Sync is selected in the method.

Note: In LC Sync mode, the mass spectrometer coordinates with the action of the LC system to make sure
that data is acquired properly.

Figure 7-3 Acquisition Method with LC Sync Selected

3. Make sure that the ion source and gas parameters are set to reasonable levels to prevent the contamination
of the mass spectrometer while optimizing. For more information, refer to the ion source Operator Guide.
4. Set the horizontal micrometer to 5.

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5. Set the vertical micrometer on the ion source for the flow rate. As a starting point, use the parameters in the
following table: For more information, refer to the ion source Operator Guide.

Table 7-8 Ion Source Vertical Parameters

Flow rate Initial Vertical Parameters

1 μL/min to 20 μL/min 10 mm
20 μL/min to 250 μL/min 5 mm
250 μL/min to 500 μL/min 2 mm
500 + μL/min 0 mm

6. Set the values for the HPLC system and use an autosampler injection volume of 10 µL. Use the same
concentration or lower as for the infusion experiment.

The LC pumps must be configured for an isocratic run with no column. The MS and LC times must be the same
to collect the proper data.
The flow rate and percent of mobile phases used should be based on the LC column used, the general
chromatography, and the approximate mobile phase concentration at which the compounds of interest elute.
7. On the Navigation bar, under Tune and Calibrate, double-click Compound Optimization.
8. On the Instrument Settings page, depending on the LC system being used, type the appropriate parameter
values. Refer to Table 7-9.

Table 7-9 Example Instrument Settings Parameters

Parameter Value
Inlet FIA
Default Acq. Method FIA_AutoOpt_Tutorial.dam
Rack Code Autosampler specific
Rack Position Autosampler specific
Injection Volume 10 µL
Mass Spectrometer MS/MS Analysis

9. Click Next.
10. Make sure that the Int. Std. check box is cleared.

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Selecting the check box indicates which MRM corresponds to the internal standards. Internal standards are
not optimized during the optimization process.
11. In the Resolution section, select Unit in both the Q1 Resolution and Q3 Resolution fields.

Figure 7-4 Q1 and Q3 Resolution Fields

12. Click Next.

13. On the FIA Source Parameters page, type numbers that are lower or higher than the original value as long as
they are still within specifications.

Be sure not to go too low with any of the settings to keep the system clean. Refer to Table 7-10 for parameters
that can be used as a starting point.

Tip! Type the values before selecting the check box.

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Automatic Optimization

Table 7-10 Example Parameters for FIA Source Parameters Page

Parameter Select the Values for Optimization

Optimize
Check Box?
Curtain Gas Yes 20;40;55
Collision Gas No —
IonSpray Voltage Yes 1500;2000;3000;4000;5000
Temperature Yes 300;400;500;600;700
Ion Source Gas 1 Yes 40;50;60;70;80;90
Ion Source Gas 2 Yes 40;50;60;70;80;90
Interface Heater No —

14. Select 1 or 2 beside Replicate Injection for each Parameter.

The total number of injections and the total sample volume are calculated based on the specifications here.
Note the total sample volume needed. Sample volume might be high depending on how many variables for
each parameter are being optimized, as each variable is a separate method.

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Figure 7-5 Example Replicate Injection for each Parameter Field

15. Click Next.

16. On the FIA Compound Parameters page for each analyte, use the example parameter values provided as a
starting point. Refer to Table 7-11.

Note: The values in Table 7-11 are suggested values. For more information, refer to the Help.

Table 7-11 Example FIA Compound Parameters Page

Parameter Select the Values for Optimization

Optimize Check
Box?
Declustering Potential Yes 60;80;100;120;200
Entrance Potential No —

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Automatic Optimization

Table 7-11 Example FIA Compound Parameters Page (continued)

Parameter Select the Values for Optimization

Optimize Check
Box?
Collision Energy Yes 20;30;40;50;70;80;100
Collision Cell Exit Yes 2;4;6;8;10;12
Potential

The total number of injections and dependent sample volume update automatically. In contrast to ion source
parameters, which require one injection per value per replicate, compound-dependent parameters only require
one injection per parameter. A looped experiment is performed for each parameter. The values are alternated
scan-by-scan within one injection.

Note: Do not enter too many values. Doing so will prevent proper evaluation of the parameter.

17. Using the Compound list, move to another compound and type the parameters to be optimized.
18. Repeat step 19 until all of the parameters for all of the compounds are provided.
19. Type 1.5 in the Mass Spec. Duration field.

Note: This value should be at least the required length of time for each injection.

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Figure 7-6 Example Mass Spec. Duration Field

20. Click Finish to begin the optimization process.

The software optimizes the specified ion source- and compound-dependent parameters to get the maximum
sensitivity for the MRM transition of the compound. As the software proceeds through the optimization, it
creates a Compound Optimization report.
21. To obtain optimized parameters, repeat this routine.

Note: Typically, the ion source and gas parameters must be narrowed using one more FIA cycle.

22. Open the final optimized FIA method called *_FIA_sample_1.

Note: The software generates several acquisition methods.

23. Save this method using a simpler name.

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Operating Instructions —
Acquisition Methods 8
An acquisition method consists of experiments and periods. Use the Acquisition Method Editor to create a sequence
of periods and experiments for the instrument and devices.
We recommend that only users who are proficient in method development create or modify acquisition and
quantitation methods. Refer to the Laboratory Director’s Guide for more information about roles and security.

Create an Acquisition Method Using the Acquisition

Method Editor
Tip! If users are creating a new acquisition method file from an existing file, then some or all of the peripheral
device methods in the acquisition method might be used.

Only devices configured in the active hardware profile appear in the Acquisition method pane. Any devices added
to the hardware profile must also be added to existing acquisition methods. For more information about devices,
refer to the Peripheral Devices Setup Guide.

1. Make sure that a hardware profile containing the mass spectrometer and peripheral devices is active.
2. On the Navigation bar, under Acquire, double-click Build Acquisition Method.
3. Select a Synchronization Mode on the Acquisition Method Properties tab.
4. (Optional) Select the Auto-Equilibration check box and then type the required equilibration time, in minutes.
5. Click the Mass Spec icon in the Acquisition method pane.
6. Select a Scan type on the MS tab.
7. Type values in the fields as required. Refer to Parameters on page 28.
8. On the Advanced MS tab, type values in the fields as required.
9. On the MS tab, click Edit Parameters.
10. On the Source/Gas tab, specify values in the fields as required.
11. On the Compound tab, specify values in the fields as required and then click OK.
12. Click a device icon and then select the parameters for the device.
13. Add any additional periods and experiments. Refer to Add an Experiment on page 85 and Add a Period on
page 86.
14. Click File > Save.

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Configure the Integrated Syringe Pump

1. Confirm that the integrated syringe pump is selected in the hardware device profile.
2. Click the Syringe Pump icon in the Acquisition method pane.

The Syringe Pump Method Properties tab opens in the Acquisition Method editor.

Figure 8-1 Syringe Pump Properties tab

3. Type the syringe diameter in the Syringe Diameter (mm) field.

4. Type the flow rate in the Flow Rate field.
5. Select the units of flow from the Unit list.

Add an Experiment
1. In the Acquisition method pane, on the period where the experiment is to be added, right-click and then click
Add experiment.

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Operating Instructions — Acquisition Methods

Figure 8-2 Add Experiment

An experiment is added below the last experiment in the period.

Note: An experiment cannot be inserted between experiments, IDA criteria, or periods. Users can only add
an experiment at the end of the period.

2. In the MS tab, select the appropriate parameters.

Add a Period
• In the Acquisition method pane, right-click the Mass Spec icon, and then click Add period.

A period is added below the last period created.

Note: Users cannot use multiple periods in an IDA experiment.

Copy an Experiment into a Period

1. Open a multi-period method.
2. In the Acquisition method pane, press Ctrl, and then drag the experiment to the period.

The experiment is copied below the last experiment in the period.

Copy an Experiment within a Period

Use this procedure to add the same or similar experiments to a period if most or all of the parameters are the
same.
• Right-click the experiment and then click Copy this experiment.

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A copy of the experiment is added below the last experiment created. This is useful when the same or similar
experiments are added to an acquisition method.

Scan Techniques
MS: In MS scans, also referred to as single MS scans, ions are separated according to their mass-to-charge ratio
(m/z). A single MS scan might be used to determine the molecular weight of a compound. Single MS scans can
also be referred to as survey scans. MS scans do not provide any information about the chemical make-up of the
ions other than the mass. Perform MS/MS or MS/MS/MS scans to obtain more information about the ions.
MS/MS: MS/MS scans are used to determine a molecular species.
• For MS/MS scans in triple quadrupole systems, precursor ion fragmentation occurs in the collision cell.
®
• For MS/MS scans in QTRAP systems, precursor ion fragmentation can occur in the collision cell or the linear
ion trap.

If enough energy is used, then the precursor ion fragments to produce characteristic product ions.
MS/MS/MS: The linear ion trap (LIT) system MS/MS/MS scans go one step further than MS/MS scans. A fragment
that is produced in the collision cell is fragmented further in the linear ion trap to give more structural information
about the molecular ion.

Quadrupole-Mode Scan Types

Triple quadrupole instruments have high-sensitivity Multiple Reaction Monitoring (MRM) capabilities required for
quantitation experiments. In addition, they have highly specific scan types, such as precursor ion and neutral loss
scans, which allow a more advanced search to be performed on the components of the samples.
Q1 MS (Q1): A full scan type using the first quadrupole (Q1). The ion intensity is returned for every mass in the
scan range.
Q1 Multiple Ions (Q1 MI): A zero-width scan type using the Q1 quadrupole. The ion intensity is returned for
the specified masses only.
Q3 MS (Q3): A full scan type using the third quadrupole (Q3). The ion intensity is returned for every mass in the
scan range.
Q3 Multiple Ions (Q3 MI): A zero-width scan type using the Q3 quadrupole. The ion intensity is returned for
the specified masses only.
MRM (MRM): An MS/MS scan in which a user-selected ion is passed through the Q1 quadrupole and then
fragmented in the Q2 collision cell. The Q3 quadrupole then selects the fragment ion that enters the detector. This
scan mode is used primarily for quantitation.
Product Ion (MS2): An MS/MS full scan where the Q1 quadrupole is fixed to transmit a specific precursor ion
and the Q3 quadrupole scans a defined mass range. Used to identify all of the products of a particular precursor
ion.

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Operating Instructions — Acquisition Methods

Precursor Ion (Prec): An MS/MS scan where the Q3 quadrupole is fixed at a specified mass-to-charge ratio to
transmit a specific product ion and the Q1 quadrupole scans a mass range. Used to confirm the presence of a
precursor ion or, more commonly, to identify compounds sharing a common product ion.
Neutral Loss (NL): An MS/MS scan where both the Q1 quadrupole and the Q3 quadrupole scan a mass range,
a fixed mass apart. A response is observed if the ion chosen by the Q1 quadrupole fragments by losing the neutral
loss (the fixed mass) specified. Used to confirm the presence of a precursor ion or, more commonly, to identify
compounds sharing a common neutral loss.

LIT-Mode Scan Types

The LIT-mode scans use the Q3 quadrupole as a linear ion trap. Ions are trapped and stored in the Q3 quadrupole
before being scanned out, giving increased sensitivity. In addition, MS/MS/MS analysis can be performed in the
linear ion trap, providing more information about the sample. LIT-mode scan types are typically used for qualitative
measurements.

Enhanced MS (EMS): Ions are scanned in the Q1 quadrupole to the linear ion trap where they are collected.
These ions are scanned out of the Q3 quadrupole to produce single MS type spectra.
Enhanced Multi-Charge (EMC): This scan type is similar to the EMS scan except that before ions are scanned
out of the linear ion trap, there is a delay period during which low-charge state ions (primarily singly-charged ions)
are allowed to preferentially escape from the linear ion trap. When the retained ions are scanned out, the
multiply-charged ion population dominates the resulting spectrum.
Enhanced Product Ion (EPI): This scan type is used to obtain a high quality MS/MS spectrum on a specific
ion. The fragmentation is done in the Q2 collision cell and thus provides the information-rich MS/MS spectrum
typical of collisionally activated dissociation (CAD) fragmentation. In this scan mode, the precursor ion to be
fragmented is first selected in the Q1 quadrupole with a mass window that is 1 Da to 4 Da wide, filtering out all
other ions. The precursor ion is fragmented by CAD gas in the Q2 collision cell. The fragment ions generated are
captured in the linear ion trap and then scanned out at one of three scan speeds, depending on the required
fragment ion resolution.
For IDA experiments, the Product Of field is set to 30 Da by default, and this value should not be changed.
Enhanced Resolution (ER): This scan type is similar to the EMS scan except that a small 30 Da mass around
the precursor mass is scanned out of the linear ion trap at the slowest scan rate to produce a narrow window of
the best-resolved spectra.
MS/MS/MS (MS3): A precursor ion is selected by the Q1 quadrupole and fragmented with collisionally activated
dissociation in the Q2 collision cell. The resulting product ions are all transmitted to the linear ion trap, where a
single product ion is then isolated. The isolated ion is further fragmented in the linear ion trap, and the resulting
product ions are scanned out of the trap at one of three scan speeds. As with any in-trap Collision Induced
Disassociation (CID) technique, there is a low mass cut-off for the second MS/MS step due to the condition that
®
the lowest mass fragment and precursor must be simultaneously stable in the trap. For QTRAP systems, this
results in the loss of ions lower than 28 percent of the mass of the precursor ion during MS3 experiments. This
phenomenon is often referred to as the one-third cut-off rule.

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About Spectral Data Acquisition

Spectral data can be acquired in one of the modes described in Table 8-1.

Table 8-1 Spectral Data

Mode Description
Profile The preset value is 0.1 Da. Profile data is the data generated by the mass
spectrometer and corresponds to the intensity recorded at a series of evenly
spaced discrete mass values. For example, for a mass range from 100 Da to
200 Da and a step size of 0.1 Da, the mass spectrometer scans 99.95 to 100.05
(recorded as value 100), 100.05 to 101.15 (recorded as value 101)…199.95
to 200.05 (recorded as value 200).
Peak Hopping The preset value is 1.0 Da. Peak Hopping is a mode of operating a mass
spectrometer in which large steps (approximately 1 Da) are made. It has the
advantage of speed (fewer data steps are made) but with the loss of peak
shape information.
Centroid The mass spectrometer scans as in Profile mode, but creates a centroid of the
data, replacing found peaks with the intensity-weighted center of gravity for
each peak. Centroid data has the advantage of significantly reducing file size.
The disadvantage is that peak shape information is lost and if data has been
collected as a centroid then it cannot be altered. We recommend the use of
profile mode and centroiding of the data post-acquisition.

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Operating Instructions — Analyze
and Process Quantitative Data 9
®
This section describes how to use the Analyst software to analyze and process quantitative data. Data can also
TM TM
be processed using the MultiQuant software. We suggest that the MultiQuant software is used to quantitate
TM
data. Refer to the documentation that comes with the MultiQuant software.
Use the sample files found in the Example folder to learn how to select samples for quantitation, how to select
preset queries and create table-specific queries, and how to analyze the acquired data. For more information about
the following topics, refer to the Advanced User Guide.
• Metric Plots
• Layout of a Results Table

Quantitative Analysis
Quantitative analysis is used to find the concentration of a specific substance in a sample. By analyzing an unknown
sample and comparing it to other samples containing the same substance with known concentrations (standards),
the software can calculate the concentration of the unknown sample. The process involves creating a calibration
curve using the standards and then calculating the concentration for the unknown sample. The calculated
concentrations of each sample are then available in a Results Table.

Quantitation Methods
A quantitation method is a set of parameters used to generate peaks in a sample. The quantitation method can
include parameters used to locate and integrate peaks, generate standard curves, and calculate unknown
concentrations. A previously saved quantitation method can be selected from the Quantitation menu in the batch.
The user can create a quantitation method before data acquisition and then apply the method to the quantitative
data automatically after the batch is complete. Alternatively, a quantitation method can be created and applied
post-acquisition.
Three tools can be used to create a quantitation method: the Quantitation Wizard, the Build Quantitation Method,
and Quick Quant.

Quantitation Wizard
With the Quantitation Wizard, a Results Table is generated at the same time as the quantitation method.
Alternatively, an existing quantitation method can be used to quantitate different sets of data.

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Build Quantitation Method

The Build Quantitation Method does not generate a quantitation Results Table although the method can
subsequently be used in the Quantitation Wizard to create a Results Table. The Build Quantitation Method can
also be used to change existing quantitation methods. This is the most flexible way of creating a quantitation
method. Refer to Create a Method Using the Quantitation Method Editor on page 91.

Quick Quant
Quick Quant is not recommended for quantitation of results.
Quick Quant is part of the Batch Editor. Use Quick Quant to add compound concentrations prior to data acquisition.
Because a sample has not been acquired, a representative sample cannot be selected nor can peaks be reviewed.
With this process, only the method components are defined.
To use a previously saved quantitation method, select it from the Quantitation menu in the batch. For instructions
on creating a batch, refer to Create and Submit a Batch on page 136.

About Results Tables

Results tables summarize the calculated concentration of an analyte in each unknown sample based on the
calibration curve. Results tables also include the calibration curves as well as statistics for the results. The user
can customize the results tables and view the results tables in layouts.
The data from a results tables can be exported to a txt file for use in other applications, such as Microsoft Excel.
The user can also export data in the table or just the data in the visible columns.

Quantitation Methods and Results Tables

For the following procedures, use the sample data that is installed in Example folder. The Triple Quad folder
contains the data files, Mix_Batch_1 and Mix_Batch_2. These sample files are used to demonstrate the usefulness
of metric plots to isolate problematic samples. The ions scanned were reserpine (609.3/195.0), minoxidil
(210.2/164.2), tolbutamide (271.1/91.1) and rescinnamine (635.3/221.2), which is the internal standard.
Mix_Batch_1 contains no errors in terms of sample preparation, whereas Mix_Batch_2 contains a QC sample
where the internal standard was added twice (sample QC2).

Create a Method Using the Quantitation Method Editor

Prerequisites
• Select the project that contains the data to be quantified.
• Switch Between Projects and Subprojects on page 64

1. Make sure that the Example folder is selected.

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2. On the Navigation bar, under Quantitate, double-click Build Quantitation Method.

The Select Sample dialog opens.

3. Double-click the Triple Quad folder in the Data Files list.
4. Select Mix_Batch_2. wiff.

The samples in the selected data file are shown in the Samples list.

Note: If the Compound ID field was populated for the samples and internal standards in the acquisition
method, then in the Internal Standards table, when a value is selected in the Q1/Q3 field, the Name
field is automatically populated.

5. Select a sample that provides a detectable signal to select integration parameters that fit the entire batch and
then click OK.
6. In the Internal Standards table, in the Name column, select rescinnamine. In Q1/Q3 column, select
635.3/221.2.
7. In the Analytes table, do the following:

a. In the Name column, select minoxidol for the Q1/Q3 column masses of 210.2/164.188, tolbutamide
for 271.3/91.146, and reserpine for 609.4/195.039.
b. In the Internal Standard column, from the list, select the rescinnamine as internal standard to be
associated with each analyte.
c. Delete 635.4/221.185 from the Q1/Q3 column in the Analytes table.

Note: If the Compound ID field was populated for the samples and internal standards in the acquisition
method, then in the Analytes table, the Name field and Q1/Q3 field are populated.

8. Click the Integration tab.

The preset integration parameters are suitable for most peaks.

9. If the integration is not suitable, then change the algorithm. Refer to Manually Integrate Peaks on page 103.
10. Click the Show or Hide Parameters icon to show the additional integration algorithms.
11. Click the Calibration tab.

The preset parameters are suitable for these samples. User can change the fit, weighting, and regression
parameter depending on the specific applications.
12. Save the quantitation method.

The new method can be used when a batch is created in the Batch Editor or when the Quantitation Wizard is
used to generate a Results Table.

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Tip! The quantitation method can only be used in the current project unless it is copied to another project.
To do this, click Tools > Project > Copy Data. A new project must be created and selected to be available
for use.

Create a Results Table Using the Quantitation Wizard

Prerequisites
• Select the project that contains the data to be quantified.
• Switch Between Projects and Subprojects on page 64

1. On the Navigation bar, under Quantitate, double-click Quantitation Wizard.

The Create Quantitation Set - Select Samples page opens.

2. Double-click the Triple Quad folder in the Available Data Files list.
3. Select Mix_batch_2. wiff.
4. Click Add All.

Note: We recommend that users do not process or report results from any sample for which acquisition was
abnormally or unexpectedly terminated.

5. Click Next.

The Create Quantitation Set - Select Settings & Query page opens.
6. Click Select Existing: Query in the Default Query section.
7. Select Accuracy 15% from the Query list.

Note: To create a query at the same time, refer to Create a Standard Query (Optional) on page 94.

Note: It is the responsibility of the user to evaluate and validate the query to be used for specific applications.

8. Click Next.

The Create Quantitation Set - Select Method page opens.

9. Click Choose Existing Method.
10. Select PK Data_Mix.qmf from the Method list.

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11. Click Finish.

The Results Table opens.

Tip! To add or remove samples in the Results Table, click Tools > Results Table > Add/Remove
Samples.

12. Review the sample type, actual concentration, peak integration, calibration curves, statistics pane, metric plot
for the internal standard, and other information related to data quantitation.
13. Save the Results Table.

Note: We recommend that users do not change datafile (wiff) names if a Results Table includes samples
from that file.

Tip! Well-formatted reports can be created from a Results Table using the Reporter software. We recommend
that the user validate the results if a Reporter template that contains a query is used. Refer to Reporter
Software on page 130.

Create a Standard Query (Optional)

Advanced users can create a query and a standard query numerous ways. The following is one example. For more
information about creating queries, refer to the Help.
1. On the Navigation bar, under Quantitate, double-click Quantitation Wizard.
2. Select samples in the Create Quantitation Set - Select Samples page.
3. Click Next.
4. In the Select Settings & Query page, in the Default Query section, select Create New Standard Query.
5. Type a query name.

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Figure 9-1 Create Quantitation Set — Select Settings & Query Page

6. Click Next.

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Figure 9-2 Create Quantitation Set — Create Default Query Page

7. In the Maximum Allowed Accuracy Variation for QCs (%) table in the Max. Variation column,
type the maximum allowable percent of variation for each QC, for example 5 is ± 5%, in the same row as the
corresponding concentration. If the concentrations were not specified during acquisition, then they are not
shown here. In this case, type them in the Concentration column.
8. In the Maximum Allowed Accuracy Variation for Standards (%) table, in the Max. Variation
column, type the maximum allowable percent of variation for each standard, for example 10 is ±10%, in the
same row as the corresponding concentration. If the concentrations were not specified during acquisition, then
they are not shown here. Type the concentrations in the Concentration column.
9. Click Next.

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Figure 9-3 Create Quantitation Set — Select Method Page

10. Select or create a method.

11. Click Finish.

The query is applied as a standard query. The query results are shown as a Pass or Fail entry in the Standard
Query Status column of the Results Table.

Tip! To return to the full view, right-click and then click Full.

Results Table Right-click Menu

Right-click in the Results Table to access the options shown in Table 9-1.

Table 9-1 Results Table Right-click Menu

Menu Function
Full Shows all the columns.
Summary Shows specific columns.
Analyte Shows a specific analyte.

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Table 9-1 Results Table Right-click Menu (continued)

Menu Function
Analyte Group Creates an analyte group.
Sample Type Shows samples of a specific type or all samples.
Add Formula Adds a formula column.
Column
We recommend that the user validate the results if a formula column is used.
Table Settings Edits or selects a table setting.
Query Creates or selects a query.
Sort Creates a sort or sorts by index.
Metric Plot Creates a metric plot.
Delete Pane Deletes the active pane.
Fill Down Copies the same data into the selected cells.
Add Custom Adds a custom column.
Column
Delete Custom Deletes the selected custom column.
Column

Peak Review and Manual Integration of Peaks

Use peak review to survey the peaks that the software has identified and then redefine the peak or the start and
end points where required.
After identifying the analytes and internal standards that the software must find, the software searches for the
peaks in the samples. When the software identifies a peak, it shows the chromatograms for each analyte and
internal standard in the Create Quantitation Method: Define Integration page of the Standard Wizard or on the
Integration tab of the Full Method Editor. The user can confirm the peaks that are found or change the quantitation
method to better define the peaks. We recommend that users manually review all integration results.

Review Peaks
During peak review, the user might want to view a peak in its entirety or to examine the baseline to find out how
well the software found the start and end points of the peak. The automatic zooming feature can be used to do
either.
To help the software find a peak, define the exact start and end points of the peak and background manually.
These changes will apply only to that individual peak unless the global method is updated.

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Note: We recommend that manually integrated results be validated.

Tip! To review an individual peak, right-click on a point on the curve and then click Show Peak. The software
opens the Peak Review window with the selected peak.

1. Right-click in the Results Table and then click Analyte.

2. Select an analyte.
3. Click Tools > Peak Review > Pane.

The peaks are shown below the Results Table with only the peaks listed in the Results Table.

Figure 9-4 Peak Review

4. Right-click in the pane and then click Options.

5. In the Peak Review Options dialog, in the Appearance section, change Num. rows to 1 and Num.
columns to 2.
6. In the Automatic Zooming section, click Zoom Y axis to: 100% of largest peak to show the entire
peak.

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Figure 9-5 Peak Review Options Dialog

Item Definition
1 Number of rows
2 Number of columns
3 Zoom Y-axis to 100% of largest peak to show the entire peak

7. Click OK.
8. To move through the peaks, click the right-pointing arrow. Refer to Figure 9-6 and Figure 9-7 on page 102.
9. Go to the second injection of standard 3.

In this example, the peak can be integrated closer to the baseline by selecting the Specify Parameters
option.

Tip! To move to a specific peak in the Peak Review pane, select the corresponding row in the Results Table.

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Figure 9-6 Peak Review Pane

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Figure 9-7 Peak Review Pane

Item Definition
1 Arrows: Click to move through the peaks.
2 Show or Hide Parameters: Click to show the integration parameters.
3 Integration parameters: Click to change the parameters.
4 Noise Percentage: Type a noise percent.
5 Apply: Click to integrate the parameters.

10. Click Show or Hide Parameters twice.

11. Click Specify Parameters - MQ III.
12. Change the Noise Percent value.
13. Click Apply.

The peak is integrated closer to the baseline.

14. If the change does not improve the peak integration, then adjust the Noise Percent parameter until the
optimal value is found.

Note: The Update Method function only updates the algorithm values for that specific analyte (or internal
standard) and not all analytes.

15. To update the algorithm for all peaks, right-click in the pane and then click Update Method.

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Figure 9-8 Update Method

Manually Integrate Peaks

Manually integrating peaks should be done last, to limit person-to-person variability. 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.

Note: Peaks that are manually integrated, or where the algorithm was changed for only that peak, are identified
in the Record Modified column of the Results Table, as are peaks that have algorithm parameter changes for a
sample that are not applied to the entire analyte group.

1. In the Peak Review pane, click Manual Integration Mode.

Figure 9-9 Peak Review Pane: Manual Integration

2. Zoom in on the lower 10% of the peak.

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Figure 9-10 Peak Review Pane: Zooming in on a Peak

3. Move the cross-hair to where the start of the peak is to be defined and then drag the cross-hair to where the
end of the peak is to be defined.

The software shades the area bounded by the base and sides of the peak. Peak parameters are gray as they
are no longer applicable because the peak was drawn manually.
4. Do one of the following:

• To make this change permanent, click Accept.

• To discard the changes, clear the Manual Integration check box.

Tip! If a peak was correct as originally selected, right-click the peak and then click Revert to Method.

Peak Review Right-Click Menu

Right-click in the Peak Review window or pane to access the options shown in Table 9-2.

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Table 9-2 Peak Review Right-click Menu

Menu Function
Options Opens the Peak Review Options dialog.
Sample Annotation Opens the Sample Annotation dialog.
Save Active to Text Saves the selected peak as a text file.
File
Show First Page Goes to the first sample.
Show Last Page Goes to the last sample.
Slide Show Peak Opens the slide show.
Review
Update Method Updates the algorithm for all peaks.
Revert to Method Selects a redefined peak based on the current quantitation method.
Delete Pane Deletes the active pane.

Calibration Curves
Use calibration curves to find the calculated concentration of samples, including quality control (QC) samples. QC
samples are added to a batch to estimate the data quality and accuracy of standards in the batch. QC samples
have known analyte concentrations but are treated as unknowns so that the measured concentrations can be
compared to the actual value.
The calibration curve is generated by plotting the concentration of the standard versus its area or height. If an
internal standard is used, then the ratio of the standard concentration or internal standard versus the ratio of the
standard peak height or area to the internal standard peak height or area is plotted. The area or height ratio of a
sample is then applied to this curve to find the concentration of the sample, as shown in the Results Table. A
regression equation is generated by this calibration curve according to the regression that was specified. The
regression equation is used to calculate the concentration of the unknown samples.

View Calibration Curves

The user can view the calibration curve and change the regression options in an open Results Table. If two or more
Results Tables are open, then the calibration curves can be overlaid. To overlay curves, make sure that the method
used to create the tables is the same.
Plot a calibration curve to see the curve used for regression. The Calculated Concentration field in the Results
Table reflects any changes resulting from the fit of the curve to the points of the standard.

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Note: This option is available only when a Results Table is open in the workspace.

1. Open a Results Table.

2. Click Tools > Calibration > Pane.

The Calibration Curve pane containing the calibration curve opens.

Figure 9-11 Calibration Curve

3. If there is more than one analyte, then use the following steps to view the calibration curve for another analyte:

a. From the Analyte list, select an analyte.

b. If required, from the next list, select Area or Height.

4. To change the regression options for the calibration curve, do the following:

a. Click Regression.

Figure 9-12 Regression Options Dialog

b. Select Linear in the Fit list.

c. Select 1 / x in the Weighting list.
d. Click OK.

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The calibration curve opens. The user can review individual peaks on the curve or exclude points from the curve
to produce a better curve.
5. If required, repeat these steps to create a more appropriate curve.
6. To save the changes, click Accept.

Overlay Calibration Curves

Tip! To examine the curve for one table more closely, right-click the curve and then click Active Plot. Select
the curve to be plotted on top.

1. With two or more open Results Tables, view a calibration curve for one of the tables.
2. Right-click the calibration curve and then click Overlay.

Figure 9-13 Overlay Dialog

3. Select the tables to overlay with the current curve.

4. Click OK.

The software plots the curves for all selected tables on the same graph.

Calibration Curve Right-Click Menu

Right-click in the Calibration window or pane table to access the options shown in Table 9-3.

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Table 9-3 Calibration Curve Right-click Menu

Menu Function
Exclude (Include) Right-click the point and then click Exclude to exclude the point from the curve.
Right-click the point and then click Include to include the point.
Exclude All Analytes (Include Right-click a point and then click Exclude All Analytes to exclude all the
All Analytes) analytes from the curve. Right-click a point and then click Include All
Analytes to include the points.
Show Peak Reviews an individual peak.
Overlay Overlays two graphs.
Active Plot Determines which plot is active.
Legend Shows the graph legend.
Log Scale X Axis* Uses a log scale for the X-axis.
Log Scale Y Axis* Uses a log scale for the Y-axis.
Delete Pane Deletes the active pane.
Home Graph Scales the graph to its original size
* A log scale arranges the data points in a more manageable view so that the effect of all points can be monitored
simultaneously. For this view, select Log Scale Y Axis versus Log Scale X and not just the log of one axis.

Sample Statistics
Use the Statistics window to view the statistics samples, typically for standards and QCs (quality controls). The
data from each available batch in the Results Table opens in tabular form in the grid and a row of data is shown
for each standard or QC concentration.

View the Statistics for Standards and QCs

When more than one Results Table is open, statistical information about the standards and QCs for additional
batches in the Statistics window can be obtained. This facilitates comparison of results between batches and
identification of trends in the standards or QCs.
1. Open a Results Table.
2. Click Tools > Statistics.
3. Select Concentration from the Statistics Metric list.
4. Select an analyte in the Analyte Name field.

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5. Select Standard in the Sample Type field.

The results are shown.

6. Look at the %CV and Accuracy columns.

The %CV shows the coefficient of variance between the measurements of a single parameter, for example
the area. Accuracy shows how close the plotted point is to the interpolated value.
7. If required, select the Display Low/High values check box and then examine the Low, High values, and
Mean for each row in the grid. Each row represents standards that have the same concentration levels.
8. Select another analyte.

The results are shown on a per-analyte basis.

9. To check for Quality Control variations at the same concentration levels, select QC in the Sample Type
field.

Compare Results Between Batches

When more than one Results Table is shown, obtain statistical information about the standards and QCs for
additional batches in the Statistics window. Normally results are compared between batches to look for trends in
the standards or QCs or to verify that the method is valid.
For two or more open Results Tables, compare results in the Statistics window. Both sets of statistics are shown
in the Statistics window.
The number of analytes and the analyte names must be the same for the data to be combined in the Statistics
pane.
1. Open a Results Table.
2. Click Tools > Statistics.
3. Do one of the following:

• To arrange the results by Results Table, select Group By Batch in the Conc. as Rows list.
• To arrange the results in order of concentration, select Group By Concentration in the Conc. as
Rows list.
• To arrange the results in order of concentration without a row showing the statistics for each group or
batch, select Group By Concentration (no All) in the Conc. as Rows list.

The software sorts the results. At the end of each batch or group, one or two additional rows are shown: All
(statistics for all Results Tables in that group) and Average (statistics on the statistics for that batch or group).

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Operating Instructions — Analyze
and Explore Data 10
Use the sample files installed in the Example folder to learn how to view and analyze data using the most common
analysis and processing tools. For more information about the following topics, refer to the Advanced User
Guide:
• Labeling graphs
• Overlaying and summing spectra or chromatograms
• Performing background subtractions
• Smoothing algorithms
• Working with smoothed data
• Working with centroid data
• Working with contour plots
• Working with the fragment interpretation tool
• Working with library databases and library records

Open Data Files

Tip! To turn off the automatic update on the mass spectrum, right-click the mass spectrum and then click Show
Last Scan. If there is a check mark beside Show Last Scan, then the spectrum will update in real-time.

1. On the Navigation bar, under Explore, double-click Open Data File.

The Select Sample dialog is shown.

2. In the Data Files list, navigate to the data file to open, select a sample, and then click OK.

The data acquired from the sample is shown. If data is still being acquired, then the mass spectrum, DAD/UV
trace, and TIC continue to update automatically.

Navigate Between Samples in a Data File

Note: If samples were saved in separate data files, then open each file individually.

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Table D-5 on page 183 shows the navigation icons used in this procedure.
• Open a data file that contains multiple samples and then do one of the following:

• Click the icon with the arrow pointing to the right to skip to the next sample in the data file.
• Click the icon with the arrow curving to the right to skip to a non-sequential sample.
• In the Select Sample dialog, from the Sample list, select the sample to view.
• Click the icon with the arrow pointing to the left to go to the previous sample in the data file.

View Experimental Conditions

The experimental conditions used to collect data are stored in the data file with the results. The information
contains the details of the acquisition method used: the MS acquisition method (that is, the number of periods,
experiments, and cycles) including instrument parameters and the HPLC device method (LC pump flow rate). In
addition, it also contains the MS resolution and mass calibration tables used for the sample acquisition. Table
10-1 shows the software functionality available when the user views the file information.

Note: If data is acquired from more than one sample into the same wiff file, then the file information pane does
not refresh automatically while scrolling through the samples. Close the file information pane and then reopen
it to view the details for the next sample in the wiff file.

• Click Explore > Show > Show File Information.

The File Information pane opens below the graph.

Tip! To create an acquisition method from the File Information pane, right-click the File Information
pane and then click Save Acquisition Method.

Table 10-1 Right-click Menu for Show File Information Pane

Menu Function
Copy Copies the selected data.
Paste Pastes data.
Select All Selects all the data in the pane.
Save To File Saves data as an rtf file.
Font Changes the font.
Save Acquisition Method Saves the acquisition method as a dam file.

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Table 10-1 Right-click Menu for Show File Information Pane (continued)

Menu Function
Save Acquisition Method to Opens the Specify Compound Information dialog. Select the IDs and molecular
CompoundDB weights to be saved in the compound database.
Delete Pane Deletes the selected pane.

Show Data in Tables

1. Open a data file.
2. Click Explore > Show > Show List Data.

The data is shown in a pane below the graph.

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Figure 10-1 Peak List Tab

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Table 10-2 Right-click Menu for Spectral Peak List Tab

Menu Function
Column Options Opens the Select Columns for Peak List dialog.
Save As Text Saves the data as a .txt file.
Delete Pane Deletes the selected pane.

Table 10-3 Right-click Menu for Chromatographic Peak List Tab

Menu Function
Show Peaks in Graph Show the peaks in two colors in the graph.
IntelliQuan Parameters Opens the Intelliquan dialog.
Save As Text Saves the data as a txt file.
Delete Pane Deletes the selected pane.

Show ADC Data

ADC (analog-to-digital converter) data is acquired from a secondary detector (for example from a UV detector
through an ADC card), and is useful for comparison with mass spectrometer data. To have ADC data available,
acquire the data and the mass spectrometer data simultaneously and then save it in the same file.
1. Make sure that the Example folder is selected.
2. On the Navigation bar, under Explore, double-click Open Data File.

The Select Sample dialog opens.

3. In the Data Files field, double-click Devices and then click Adc16chan.wiff.
4. In the Samples list, select a sample, and then click OK.
5. Click Explore > Show > Show ADC Data.

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Figure 10-2 Select ADC Channel Dialog

6. In the Channel list, select a channel, and then click OK.

The ADC data is shown in a new pane beneath the active pane.

Show Basic Quantitative Data

1. Open a data file.
2. Click Explore > Show > Show List Data.

Figure 10-3 List Data

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3. In the Peak List tab, right-click and then select Show Peaks in Graph.

Peaks are shown in two colors.

4. To change the peak finding algorithm settings, right-click and then select either Analyst Classic Parameters
or Intelliquan Parameters, whichever is active.
5. (Optional) To remove the colored peaks, right-click in the Peak List tab and then clear Show Peaks in Graph.

Chromatograms
A chromatogram is a graphical view of the data obtained from the analysis of a sample. It plots the signal intensity
along an axis that shows either time or scan number. For more information about software functionality available
for chromatograms, refer to Table 10-6 on page 125.
The software plots intensity, in counts per second (cps), on the Y-axis against time on the X-axis. Peaks above a
set threshold are labeled automatically. In the case of LC-MS, the chromatogram is often shown as a function of
time. Table 10-4 contains the a description of the types of chromatograms.
Refer to Table 10-8 on page 128 for more information about using the available icons.

Table 10-4 Types of Chromatograms

Types of Chromatograms Purpose

TIC (Total Ion Chromatogram) A chromatographic view generated by plotting the intensity of all ions in a scan
against time or scan number.
When a data file is opened, it is preset to open as a TIC. If the experiment
contains only one scan, then it is shown as a spectrum.
If the MCA check box is selected during acquisition of the data file, then the
data file opens to the mass spectrum. If the MCA check box is not selected, then
the data file opens as the TIC.

XIC (Extracted Ion An ion chromatogram created by taking intensity values at a single, discrete
Chromatogram) mass value, or a mass range, from a series of mass spectral scans. It indicates
the behavior of a given mass, or mass range, as a function of time.
BPC (Base Peak Chromatogram) A chromatographic plot that shows the intensity of the most intense ion within
a scan versus time or scan number.
TWC (Total Wavelength A chromatographic view created by summing all of the absorbance values in
Chromatogram) the acquired wavelength range and then plotting the values against time. It
consists of the summed absorbances of all ions in a scan plotted against time
in a chromatographic pane.

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Table 10-4 Types of Chromatograms (continued)

Types of Chromatograms Purpose

XWC (Extracted Wavelength A subset of TWC. An XWC shows the absorbance for a single wavelength or the
Chromatogram) sum of the absorbance for a range of wavelengths.
DAD (Diode Array Detector) A UV detector that monitors the absorption spectrum of eluting compounds at
one or more wavelengths.

Show TICs from a Spectrum

To see an example data file, make sure that the Example project is selected. Open the LIT folder, and then open
the Reserpine.wiff file.
• Click Explore > Show > Show TIC.

The TIC opens in a new pane.

Tip! Right-click inside a pane containing a spectrum and then click Show TIC.

Show a Spectrum from a TIC

A TIC is created by summing the intensity contributions of all ions from a series of mass scans. Use the TIC to view
an entire data set in a single pane. It consists of the summed intensities of all ions in a scan plotted against time
in a chromatographic pane. If the data contains results from multiple experiments, then the user can create
individual TICs for each experiment and another TIC that represents the sum of all experiments.
When a data file is opened, it is preset to appear as a TIC. However, if the experiment contains only one scan, it
is shown as a spectrum. If the user selects the MCA check box before acquiring the data file, then the data file
opens to the mass spectrum. If the MCA check box is not selected, then the data file opens with the TIC.
1. In a pane containing a TIC, select a range.
2. Click Explore > Show > Show Spectrum.

The spectrum opens in a new pane.

Tip! Double-click in the TIC pane at a particular time to show the spectrum.

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Operating Instructions — Analyze and Explore Data

Figure 10-4 Example of a TIC

About Generating XICs

XICs can be generated only from single-period, single-experiment chromatograms or spectra. To obtain an XIC
from multi-period or multi-experiment data, split the data into separate panes by clicking the triangle under the
X-axis. Refer to Table 10-8 on page 128 for more information about using the available icons.
Several methods are available for extracting ions to generate an XIC, depending on whether chromatographic or
spectral data is used. Table 10-5 contains a summary of methods that can be used with chromatograms and
spectra.

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Table 10-5 Summary of XIC Generation Methods

Method Use with Use with Extraction

Chromatogram Spectrum
Selected range No Yes Extracts ions from a selected area in a
spectrum.
Maximum No Yes Extracts ions from a selected area in a spectrum
using the most intense peak in the selected
area. This option creates an XIC using the
maximum mass from the selected spectral
range.
Base peak masses Yes Yes Can be used only with Base Peak
Chromatograms (BPCs). Use the Use Base Peak
Masses command to extract ions results in an
XIC with a different colored trace for each mass.
If the selection includes multiple peaks, then
the resulting XIC will have an equal number of
colored traces, one for each mass.
Specified masses Yes Yes Extracts ions from any type of spectrum or
chromatogram. Select up to ten start and stop
masses for which to generate XICs.

Generate an XIC Using a Selected Range

1. Open a data file containing spectra.
2. Select a range by pressing the left mouse button at the start of the range, dragging the cursor to the stop point,
and then releasing the left mouse button.

The selection is indicated in blue.

3. Click Explore > Extract Ions > Use Range.

An XIC of the selection opens in a pane below the spectrum pane. The experiment information at the top of
the pane contains the mass range and the maximum intensity in counts per second.

Generate an XIC Using the Maximum Peak

1. Open a data file containing spectra.
2. Select a range in a spectrum.

The selection is indicated in blue.

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Operating Instructions — Analyze and Explore Data

3. Click Explore > Extract Ions > Use Maximum.

An XIC of the maximum peak specified selection opens below the spectrum pane. The experiment information
at the top of the pane contains the mass range and the maximum intensity in counts per second.

Generate an XIC Using Base Peak Masses

1. Open a data file containing spectra.
2. In a BPC, select the peak from which to extract ions.

The selection is indicated in blue.

3. Click Explore > Extract Ions > Use Base Peak Masses.

An XIC of the specified selection opens below the spectrum pane. The experiment information at the top of
the pane shows the mass range and the maximum intensity in counts per second.

Extract Ion by Selecting Masses

1. Open a spectrum or chromatogram.
2. Click Explore > Extract Ions > Use Dialog.

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Figure 10-5 Extract Ions Dialog

3. Type the values for each XIC to be created. If a stop value is not typed, then the range is defined by the start
value.

• In the Start field, type the start value (lower value) for the mass range.
• In the Stop field, type the stop value (higher value) for the mass range.

4. Click OK.

An XIC of the selection opens below the chromatogram pane. The experiment information at the top of the
pane includes the masses and the maximum intensity in counts per second.

Generate BPCs
BPCs can be generated only from single-period, single-experiment data.
1. Open a data file.
2. Select an area within a TIC.

The selection is indicated in blue.

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3. Click Explore > Show > Show Base Peak Chromatogram.

The selections are shown in the Start Time and End Time fields.

Figure 10-6 Base Peak Chromatogram Options

4. In the Mass Tolerance field, type the value to indicate the mass range used to find a peak. The software
finds the peak using a value twice the typed range (± the mass value).
5. Type the intensity below which peaks are ignored by the algorithm in the Minimum Intensity field.
6. Type the mass that determines the beginning of the scan range in the Minimum Mass field.
7. Type the mass that determines the end of the scan range in the Maximum Mass field.
8. To set the start and end times, select the Use Limited Range check box and do the following:

• In the Start Time field, type the time that determines the start of the experiment.
• In the End Time field, type the time that determines the end of the experiment.

9. Click OK.

The BPC is generated in a new pane.

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Generate XWCs
An XWC is a wavelength chromatogram created by taking intensity values at a single wavelength, or by the sum
of the absorbance for a range of several wavelengths. Up to three ranges can be extracted from a DAD spectrum
to generate the XWC. Refer to Table 10-8 on page 128 for more information about using the available icons.
1. Open a data file that contains a DAD spectrum.
2. Right-click anywhere in the pane and then click Extract Wavelengths.

Figure 10-7 Extract Wavelengths Dialog

3. Type Start and Stop values.

4. Click OK.

The XWC is shown in a pane below the DAD spectrum.

Generate DAD Data

Like mass spectrometer data, DAD data can be viewed in chromatogram or spectrum form. Users can view the
DAD spectrum for a single point in time, or for a range of time as a Total Wavelength Chromatogram (TWC).
1. Open a data file containing data acquired with a DAD.

The TWC, which is analogous to a TIC, opens in a pane below the TIC.
2. In the TWC pane, click a point to select a single point in time, or highlight an area of the spectrum to select a
range of time.
3. Click Explore > Show > Show DAD Spectrum.

The DAD spectrum opens in a pane below the TWC. The Y-axis shows the absorbance and the X-axis shows
the wavelength.

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Operating Instructions — Analyze and Explore Data

Tip! If the pane with the TWC is closed, then click a point anywhere in the TWC to open it again. Click
Explore > Show > Show DAD TWC.

Generate TWCs
A TWC is a less commonly used chromatogram. It shows the total absorbance (mAU) as a function of time. The
TWC provides a way of viewing an entire data set in a single pane. It consists of the summed absorbances of all
ions in a scan plotted against time in a chromatographic pane. If the data contains results from multiple experiments,
then create individual TWCs for each experiment and another TWC that represents the sum of all experiments.
A TWC shows total absorbance (mAU) on the Y-axis plotted against time on the X-axis. Refer to Table 10-8 on
page 128 for more information about using the available icons.
1. Open a data file that contains a DAD spectrum.
2. Click Explore > Show > Show DAD TWC.

The TWC is shown in a pane below the DAD spectrum.

Tip! Right-click inside the pane containing the DAD spectrum and then click Show DAD TWC.

Adjust the Threshold

The threshold is an invisible line drawn parallel to the X-axis of a graph that sets a limit below which the software
will not include peaks in a spectrum. The line has a handle, represented by a blue triangle to the left of the Y-axis.
Click the blue triangle to view a dotted line that represents the threshold. The threshold can be raised or lowered,
but changing the threshold value does not change the data. The software does not label any peaks in the region
that lies below the threshold.
1. Open a data file.
2. Do one of the following:

• To raise the threshold, drag the blue triangle up the Y-axis. To lower the threshold, drag the blue triangle
down.
• Click Explore > Set Threshold. In the Threshold Options dialog that opens, type the threshold value
and then click OK.
• Click Explore > Threshold.

The graph updates to show the new threshold. Peak labeling and the peak list are also updated.

Tip! To view the current threshold value, move the pointer over the threshold handle.

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Chromatogram Panes
Table 10-6 Right-click Menu for Chromatogram Panes

Menu Function
List Data Lists the data points and integrates the peaks found in chromatograms.
Show Spectrum Generates a new pane containing the spectrum.
Show Contour Plot Shows a color-coded plot of a data set, where the color represents the intensity of
the data at that point. Only certain MS modes are supported.
Extract Ions Extracts a specific ion or set of ions from a selected pane and then generates a new
pane containing a chromatogram for the specific ions.
Show Base Peak Generates a new pane containing a base peak chromatogram.
Chromatogram
Show ADC Data Generates a new pane containing the ADC data trace, if acquired.
Show UV Detector Data Generates a new pane containing the UV data trace, if acquired.
Spectral Arithmetic Wizard Opens the Spectral Arithmetic Wizard.
Save to Text File Generates a text file containing the data in a pane, which can be opened in Microsoft
Excel or other programs.
Save Explore History Saves information about changes to processing parameters, also called processing
options, that were made when a wiff file was processed in Explore mode. The
processing history is stored in a file with an eph (Explore Processing History) extension.
Add Caption Adds a caption at the cursor point in the pane.
Add User Text Adds a text box at cursor point in the pane.
Set Subtract Range Sets the subtract range in the pane.
Clear Subtract Range Clears the subtract range in the pane.
Subtract Range Locked Locks or unlocks the subtract ranges. If the subtract ranges are not locked, then each
subtract range can be moved independently. The subtract ranges are preset to locked.
Delete Pane Deletes the selected pane.

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Operating Instructions — Analyze and Explore Data

Spectra Panes
Table 10-7 Right-click Menu for Spectra Panes

Menu Function
List Data Lists the data points and integrates chromatograms.
Show TIC Generates a new pane containing the TIC.
Extract Ions (Use Range) Extracts a specific ion or set of ions from a selected pane and then
generates a new pane containing a chromatogram for the specific
ions.
Extract Ions (Use Maximum) Extracts ions using the most intense peak in a selected area.
Save to Text File Generates a text file of the pane, which can be opened in Microsoft
Excel or other programs.
Save Explore History Saves information about changes to processing parameters, also
called Processing Options, that were made when a wiff file was
processed in Explore mode. The processing history is stored in a file
with an eph (Explore Processing History) extension.
Add Caption Adds a caption at the cursor position in the pane.
Add User Text Adds a text box at the cursor position in the pane.
Show Last Scan Shows the scan prior to the selection.
Select Peaks For Label In this dialog, select the parameters to reduce peak labeling.
Delete Pane Deletes the selected pane.
Add a Record Adds records and compound-related data, including spectra, to the
library. An active spectrum is required to perform this task.
Search Library Searches the library without constraints or with previously saved
constraints.
Set Search Constraints Searches the library using the criteria typed in Search Constraints
dialog.

Graphical Data Processing

Graphical data can be processed many ways. This section provides information and procedures for using some of
the most commonly used tools.

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The user can zoom in on part of a graph to view a particular peak or an area in greater detail in both spectra and
chromatograms. The user can also zoom in repeatedly to view smaller peaks.

Graphs
The same data can be examined in different ways. Data can also be kept for comparison purposes before performing
processing operations such as smoothing or subtraction.
A window contains one or more panes arranged in such a way that all the panes are fully visible and that they do
not overlap.
Panes might be of a variable or fixed size. Panes are automatically tiled within the window and are arranged into
column and row format. If the size of a window is changed, then the panes within the window change in size to
accommodate the new size. A window cannot be sized to the point where any of the panes become smaller than
their minimum size.
Two or more windows or panes containing similar data can be linked, for example, spectra with similar mass
ranges. As one pane or window is zoomed in, the other pane zooms in simultaneously. For example, the user can
link an XIC to the BPC from which the XIC was extracted. Zooming in the BPC also zooms the XIC, so that both
chromatograms show the same magnification.

Manage Data
Data can be compared or examined in different ways. Users might want to keep the data for comparison purposes
before performing processing operations such as smoothing or subtraction.
A window contains one or more panes, arranged in such a way that all the panes are fully visible and they do not
overlap.
Panes can be of variable or fixed size. Panes are automatically tiled within the window and are arranged into
column and row format. If window size is changed, then the panes within the window change in size to
accommodate the resizing. A window cannot be resized to the point where any of the panes would become smaller
than its minimum size.
Two or more windows or panes containing similar data can be linked, for example, spectra with similar mass
ranges. When the user zooms in one pane or window, the other pane zooms simultaneously. For example, the
user can link an XIC to the BPC from which it was extracted. Zooming in the BPC also zooms the XIC, so that both
chromatograms are shown with the same magnification.
• Use the following menu options or icons to manage data in graphs.

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Operating Instructions — Analyze and Explore Data

Table 10-8 Graph Options

To do this... use this menu option... ...or click this icon

Copy a graph to a new Select the graph to copy. Click Explore > Duplicate
window Data > In New Window.
Rescale a graph to its Select the graph. Click Explore > Home Graph.
original size
Move a pane • Select the graph. Click Window > Move Pane.
• Select the pane or window and then drag it to the new
position. This position can be inside the same window
or within another window.
A four-headed arrow is shown when the cursor is on the
boundary of the active window or pane.
• If the pane is at the top or bottom of the target pane,
then the pane moves above or below that pane,
respectively.
• If the pane is at the left or right of the target pane, then
the pane moves to the left or right of that pane,
respectively.
• If the pane is at any other position, then the pane
moves to the target row. The drop shadow of the pane
as the pane is moved indicates its new position.

Link panes a. With the two graphs open, click one to make that pane
active.
b. Click Explore > Link and then click the other pane.

Remove linking Close one of the panes. Click Explore > Remove Link.

Delete a pane Select the graph. Click Window > Delete Pane.

Lock a pane Select the graph. Click Window > Lock Panes.

Hide a pane Select the graph. Click Window > Hide Pane.

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Table 10-8 Graph Options (continued)

To do this... use this menu option... ...or click this icon

Maximize a pane Select the graph. Click Window > Maximize Pane.

Tile panes Select the graph. Click Window > Tile all Panes.

Zoom In on the Y-axis

1. Move the pointer to the left of the Y-axis to either side of the area to be expanded and then drag away from
the starting point in a vertical direction while holding the left mouse button.

A box is drawn along the y-axis representing the new scale.

Note: Take care when zooming in on the baseline. Zoom in too far and the zoom-in box closes.

2. Release the mouse button to draw the graph to the new scale.

Zoom In on the X-axis

Tip! To return the graph to the original scale, double-click either axis. To restore the entire graph to the original
scale, click Explore > Home Graph.

1. Move the pointer under the X-axis to either side of the area to be expanded and then drag away from the
starting point in a horizontal direction while holding the left mouse button.
2. Release the mouse button to draw the graph to the new scale.

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Reporter Software 11
®
The Reporter software extends the reporting functionality available in the Analyst software.
We recommend that users validate the results if a modified Reporter template or one that contains a query is used.
The Reporter software can be used to create custom reports with Microsoft Word and Excel (2010, 2013, or 2016).
The Reporter software has the following features:
• Provides a variety of reports that use the data available in a Results Table, in file information, and in quantitative
peak review windows.
• Provides a variety of reports that present MS/MS library search results. The user can configure the Reporter
®
software to search against any MS/MS spectral library that uses the Analyst (mdb) format.
• Uses Microsoft Word templates to provide the format information needed when generating reports. These
templates can be created or modified to provide customized report formats. Refer to the Help for information
about creating or editing the Report Template Editor.
®
• Contains a blank starting template that can be used in the Analyst software Reporter editing environment to
design report templates to meet most reporting requirements.
• Automates report generation through the use of the Autoquan Reporter batch script.
• Automatically prints, exports to Adobe Portable Document Format (pdf), and delivers results by e-mail.
• Attaches processing scripts to report templates to expand both the content and automation level for various
workflow requirements.
®
• Generates reports from custom software applications that use the available Analyst software programming
libraries.
Reporter software can be used as follows:
®
• Within the Analyst software to manually generate a report or set of reports.
• By a batch script to automate report generation within a batch. Users can generate reports on a
sample-by-sample basis, either during or after batch acquisition.
®
• By applications that do not use the Analyst software.

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 Reporter Software

Analyst Reporter User Interface

Figure 11-1 Analyst Reporter

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Reporter Software

Item Option Description

1 File > Exit Exits the program and releases all resources.
2 Settings > Select Output Language Sets the language dictionary that will be used to replace
language tags within a report template. Templates that
contain language tags can be used to generate reports
in any language. The language tags are replaced with
text from a matching tag in the dictionary file for the
selected language. These dictionary files are contained
in the folder: C:\Program Files\AB
SCIEX\AnalystReporter\Resources\Languages on
Windows 7, 32- bit operating systems or C:\Program
Files (x86)\AB
SCIEX\AnalystReporter\Resources\Languages on
Windows 7, 64-bit or Windows 10, 64-bit operating
systems.
2 Settings > Select Library Browse to a spectral library. This library will be used for
matching and scoring MS/MS data from Results Tables
that contain data from information dependent
acquisition (IDA) triggered MS/MS scan types.
2 Settings > Select Template Folder Sets the folder from which the available templates will
be read. To return to the default template folder, select
the Default option.
3 Help > About Shows information about the version of Reporter
software currently installed.
4 Current Output Language Shows the currently selected language dictionary used
for replacing language tags within a report template.
The language dictionary can be selected using Settings
> Select Output Language.
5 Current Spectral Library Shows the currently selected spectral library. The spectral
library can be selected using Settings > Select
Library.
6 Available Templates and Description Shows a list of available report templates. Selecting a
template will show a description of the template. To
change the folder from which available templates are
read, select Settings > Select Template Folder
> Browse.

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 Reporter Software

Item Option Description

7 Output Format Shows the output formats that are supported by the
Reporter software. Only formats that are compatible
with the selected report template are enabled.
• Word: Microsoft Word document (docx) is produced.
This document can be viewed by Microsoft Word
2010 and above.
• PDF: A report is created directly in PDF format.
• HTML: Microsoft Word is used to generate an HTML
file. Associated image files are stored in a folder with
the same name as the HTML file.
• Excel: A plain text file (csv) is produced. Report
templates that contain values separated by commas
can be opened in Microsoft Excel, where each value
will be shown in a separate cell. Only templates that
are specifically marked as text-compatible can be
used for this output format.
• Text: A plain text document (txt) is produced. Only
templates that are specifically marked as text
compatible can be used for this output format.

8 Print Automatically After the report has been created it is printed to the
selected printer. Select any available printer.
9 Create Report Creates the report in the selected output format using
the selected report template.
10 Close Exits the program and releases all resources.

Generate Reports
The Reporter software extracts numerical data from the Results Table and sample and graphical information from
the wiff file.
Select a template in the Available Template field.

Tip! For reports that can be generated on a sample-by-sample basis, it may be more efficient to generate the
reports automatically using a batch script during acquisition to avoid long processing times at the end of the
acquisition. For more information about batch scripts, refer to the Scripts User Guide.

1. Open a Results Table.

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Reporter Software

2. Under Companion Software, double-click Reporter.

3. In the Analyst Reporter dialog, in the Available Templates field, select the applicable report template.
4. Click the PDF output format.

The Word option is pre-selected and the report is automatically saved in the current project Results folder. If
this option is not selected, then the report is created and opened in Word or printed as selected, but the report
is not saved. This lets the user edit the report in Word prior to saving the original report.
5. Select either one document containing all samples or multiple documents with one sample in each.
6. (Optional) Select the Print Automatically check box to print the reports automatically on a pre-selected
printer.

The Default Printer set in Windows is used unless a different printer is selected. The Reporter tool retains the
selected printer between operations. If the printer is set to a PDF printer driver, then the Reporter generates
PDF file versions of the created reports automatically.
7. Click Create Report.

The screen shows various progress indicators as the tool opens the template and populates it with data from
the Results Table. Some reports can take seconds to generate, others can take longer. A large data set with
many MRM transitions or a large number of graphics might result in reports of several hundred pages that
take hours to generate.

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Operating Instructions — Batches 12
CAUTION: Potential System Damage. If the HPLC system connected to the mass spectrometer
are not controlled by the software, then do not leave the mass spectrometer unattended
while in operation. The liquid stream from the HPLC system can flood the ion source when
the mass spectrometer goes into Standby mode.

Set Queue Options

The queue goes one-by-one through the list, acquiring each sample with the selected acquisition method. After
all of the samples have been acquired, the queue stops and the mass spectrometer goes into Standby mode. In
Standby mode, the LC pumps and some instrument voltages are turned off.
®
The user can change the length of time the queue runs after the last acquisition has finished, before the Analyst
software puts the mass spectrometer into Standby mode. For information about the other fields in the Queue
Options dialog, refer to the Help.
1. On the Navigation bar, click Configure.
2. Click Tools > Settings > Queue Options.

Figure 12-1 Queue Options Dialog

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Operating Instructions — Batches

3. In the Max. Num. Waiting Samples field, set the maximum number of samples to a value that is greater
than the number of samples that will be submitted to the queue
4. In the Max. Idle Time field, type the length of time the queue will wait after acquisition is completed before
going to Standby mode. The preset value is 60 minutes.

If gas cylinders are used, then adjust this time to make sure that the gas in the cylinders is not depleted.
If an LC method is used, then before the run is started, make sure that there is enough solvent in the reservoirs
for all of the sample runs at the primary flow rate and the maximum idle time.
5. Select the Leave Mass Spec on in Standby check box to keep the mass spectrometer running after
analysis has been completed. This feature allows the heaters and gases to continue running, even after devices
have entered Idle state, so that the ion source and entrance to the mass spectrometer are kept free of
contaminants.
6. Select the Fail Whole Batch in Case of Missing Vial check box to fail the entire batch when a missing
vial is encountered. If this option is not selected, then only the current sample will fail and the queue will
continue to the next sample.

Create and Submit a Batch

Use this workflow to create a batch. In this example, use the MRM scan type that was created previously. Go
through the workflow twice more for practice, once using the Q1MS method and the second time using the Q1MI
method.

Add Sets and Samples to a Batch

A set can consist of a single sample or multiple samples.

Note: For more information about adding quantitation information to a batch, refer to the Advanced User
Guide.

1. On the Navigation bar, under Acquire, double-click Build Acquisition Batch.

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 Operating Instructions — Batches

Figure 12-2 Batch Editor Dialog

2. In the Sample tab, in the Set list, type a name.

3. Click Add Set.
4. Click Add Samples to add samples to the new set.

Figure 12-3 Add Sample Dialog

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Operating Instructions — Batches

5. In the Sample name section, in the Prefix field, type a name for the samples in this set.
6. To add incremental numbering to the end of the sample name, select the Sample number check box.
7. If the Sample number check box is selected, then in the Number of digits field, type the number of
digits to include in the sample name.

For example, if 3 is typed, then the sample names would be samplename001, samplename002, and
samplename003.
8. In the Data file section, in the Prefix field, type a name for the data file that will store the sample information.
9. Select the Set name check box to use the set name as part of the data file name.
10. Select the Auto Increment check box to increment the data file names automatically.

Note: The data for each sample can be stored in the same or a separate data file. The names of the data
file will have numerical suffixes starting from 1.

11. Type a name in the Sub Folder field.

The folder is stored in the Data folder for the current project. If the Sub Folder field is left blank, then the
data file is stored in the Data folder and a subfolder is not created.
12. In the New samples section, in the Number field, type the number of new samples to add.
13. Click OK.

The sample table fills with the sample names and data file names.

Tip! Fill Down and Auto Increment options are available in the right-click menu after a single column
heading or several rows in a column are selected.

14. On the Sample tab, in the Acquisition section, select a method from the list.

Depending on how the system is set up, specific information for the autosampler must be entered. Even if the
injection volume is set in the method, the user can change the injection volume for one or more samples by
changing the value in the injection volume column.

Note: To use different methods for some of the samples in this set, select the Use Multiple Methods
check box. The Acquisition Method column is shown in the Sample table. Select the acquisition method
for each sample in this column.

15. To change the injection volumes from the volumes listed in the method, in the Inj. Volume (µL) column,
type the injection volume for each sample.
16. To set sample locations, do one of the following:

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• Set Sample Locations in the Batch Editor on page 141

• Select Vial Positions Using the Locations Tab (Optional) on page 142

17. (Optional) To define quantitation details prior to submitting the batch, refer to Set Quantitation Details in the
Batch Editor (Optional) on page 143.
18. Click the Submit tab.

Note: The order of samples can be edited before the samples are submitted to the queue. To change the
order of samples, on the Submit tab, double-click any of the numbers at the far left of the table (a very
faint square box is shown), and then drag them to the new location.

19. If the Submit Status section contains a message about the status of the batch, then do one of the following:

• If the message indicates that the batch is ready for submission, then proceed to step 20.
• If the message indicates that the batch is not ready for submission, then make the changes as indicated by
the message.

20. After confirming that all of the batch information is correct, click Submit.

The batch is submitted to the queue and can be viewed in the Queue Manager.
21. Save the file.

Equilibrate the System

Equilibrate the system before submitting a batch. Equilibration warms up and prepares the mass spectrometer for
the next sample or batch.

1. Click .

The Equilibrate dialog opens.

2. Select the acquisition method used for the submitted batch.
3. Type the equilibration time in the Time (min) field, in minutes.
4. Select OK. The system starts the equilibration.

After the equilibration is completed, the system will change to Ready status.

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Operating Instructions — Batches

Tip! If the equilibration does not finish as expected, or if the system status does not change to Ready after
the equilibration finishes, then make sure that
• The activated hardware profile is appropriate for the acquisition method.
• The HPLC system is turned on.
• The HPLC system has correctly established communication with the software.

Submit a Sample or Set of Samples

Note: Run the sample again in the event of an abnormal termination during sample acquisition. If the abnormal
termination is caused by a power failure, then the temperature of the autosampler tray is not maintained and
sample integrity might be compromised.

1. Select one sample or a set of samples.

2. Click the Submit tab in the Batch Editor.
3. If the Submit Status section contains a message about the status of the batch, then do one of the following:

• If the message indicates that the batch is ready for submission, then proceed to the next step.
• If the message indicates that the batch is not ready for submission, then make the changes as indicated by
the message.

4. Click Submit.

Change Sample Order

The order of the samples can be edited before the samples are submitted to the Queue.
• On the Submit tab, double-click any of the numbers at the far left of the table (a very faint square box is
visible), and then drag them to the new location.

Acquire Data
The system must not be in Tune and Calibrate mode when sample acquisition is started. Also, if the system has
been previously run that day and has not yet been set to Standby mode, then sample acquisition will start
automatically.
1. Make sure that the column oven temperature is reached.

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2. Make sure that the icon is not pressed in.

3. On the Navigation bar, click Acquire.
4. Click View > Sample Queue.

The Queue Manager opens with all submitted samples.

Figure 12-4 Queue Manager

Item Description
1 The Reserve Instrument for Tuning icon should not be pressed in.
2 Queue status should be in Ready mode.
3 Queue Server should be in Normal. Refer to Queue States on page 146.

5. Click Acquire > Start Sample.

Set Sample Locations in the Batch Editor

If an autosampler is used in the acquisition method, then the vial positions of the samples must be defined in the
acquisition batch. Define the location in the Sample tab or in the Locations tab. For more information about
creating batches, refer to Add Sets and Samples to a Batch on page 136.
1. In the Sample tab, from the Set list, select the set.
2. For each sample in the set, do the following if applicable:

• In the Rack Code column, select the rack type.

• In the Rack Position column, select the position of the rack in the autosampler.
• In the Plate Code column, select the plate type.
• In the Plate Position column, select the position of the plate on the rack.
• In the Vial Position column, type the position of the vial in the plate or tray.

3. Save the file.

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Operating Instructions — Batches

Select Vial Positions Using the Locations Tab

(Optional)
1. Click the Locations tab in the Batch Editor.
2. Select the set from the Set list.
3. Select the autosampler from the Autosampler list.
4. In the space associated with the rack, right-click and then select the rack type.

The plates or trays are shown in the rack.

5. Double-click in the white space labeled rack type. A visual sample rack layout is shown.

The appropriate number of rack spaces for the autosampler is shown in the graphic rack view.
6. Double-click one of the rectangles.

The circles depicting the wells or vials for the plate or tray are shown.

Tip! To see the corresponding vial number in the graphical representation, move the cursor over the sample
position. Use this information to confirm that the vial positions in the software match the vial positions in
the autosampler.

Figure 12-5 Locations Tab

Note: Depending on the autosampler being used, it might not be necessary to type details in additional
columns.

7. To select whether samples are marked by row or column, click the Row/Column selection selector button.

If the button shows a red horizontal line, then the Batch Editor marks the samples by row. If the button
shows a red vertical line, then the Batch Editor marks the samples by column.

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8. Click the sample wells or vials in the order to be analyzed.

Tip! Click a selected well or vial again to clear it.

Tip! To fill in the samples automatically, press the Shift key while clicking the first and last vial within a
set. To perform multiple injections from the same vial, press the Ctrl key while clicking the vial location. The
red circle changes to a green circle.

Set Quantitation Details in the Batch Editor

(Optional)
If a quantitation method is used with a batch and if the user does not want to select quantitation details after
acquisition, then the quantitation details (sample type, sample concentration) must be defined before the batch
is submitted.
The appropriate Internal Standard and Standard columns are shown in the Quantitation tab according to the
quantitation method selected in the Sample tab.
1. With a batch file open in the Batch Editor window, click the Quantitation tab.
2. Select the set containing the samples.
3. Select a Quant Type for all the samples from the list in the cell.
4. If applicable, type the analyte concentration in the Analyte column.
5. If applicable, type the internal standard concentration in the Internal Standard column.
6. Repeat this procedure for each set in the batch.

Stop Sample Acquisition

When a sample acquisition is stopped, the current scan finishes before the acquisition is stopped.
1. In the Queue Manager, click the sample in the queue after the point where acquisition should stop.
2. On the Navigation bar, click Acquire.
3. Click Acquire > Stop Sample.

The queue stops after the current scan in the selected sample is complete. The sample status in the Queue
Manager (Local) window changes to Terminated, and all other samples following in the queue are
Waiting.
4. To continue processing the batch, click Acquire > Start Sample.

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Operating Instructions — Batches

Import Batch Files

Users can import a text file containing batch information instead of creating a batch in the Batch Editor. If all
the sample details are in a spreadsheet, then it is faster to rearrange and import the data in the spreadsheet than
to manually type the data into the Batch Editor.
Before importing batch information from a text file, make sure that the data in the file is organized and formatted
correctly. In particular, the column headings in the spreadsheet must match the Batch Editor column headings.

Build a Batch as a Text File

Prerequisites
Make sure that the active hardware profile includes all of the devices to be used to acquire the samples.
To make sure that the text file includes the proper headings, create a batch using the Batch Editor, export it as a
text file, type the appropriate values in a spreadsheet editor, and then import the file back into the Batch Editor.
Users can export a batch only if it contains at least one set with at least one sample. The saved text file can be
used again later as a template.
1. In the Batch Editor, create a single-set, single-sample batch.
2. Click File > Export.

The Save As dialog opens.

3. Type a name for the text file in the File name field and then click Save.
4. Open the text file in a spreadsheet program such as Microsoft Excel.
5. Type, or copy and paste, the details for the samples: one sample per row, with the details under the appropriate
headings.

Note: Do not delete any of the columns. The columns in the spreadsheet must match the columns in the
Batch Editor.

6. Save the modified text file as a .txt or .csv file and then close the spreadsheet program.

The text file is now ready to be imported into the Batch Editor.

Import a Batch from a Text File

1. In the Batch Editor, in the Sample tab, right-click, and then click Import From > File.

The Open dialog opens.

2. Click the required text file and then click Open.

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If an autosampler is used, then the Select Autosampler dialog opens.

Note: If the saved text file is not visible in the Files of type list, then select Microsoft Text Driver
(*.txt; *.csv). Files with the extension .txt are shown in the field.

3. In the autosampler list, select the autosampler and then click OK.

The sample table fills with the details from the text file.
4. Submit the batch.

Batch Editor Right-click Menu

Right-click in the Batch Editor table to access the options.

Figure 12-6 Batch Right-Click Menu

Menu Function
Open Opens a batch file.
Import From Imports a file.
Save As Batch Saves the batch with a different name.
Save As a Template Saves the batch as a template.
Hide/Show Column Hides or shows a column.

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Operating Instructions — Batches

Menu Function
Save Column Settings Saves the batch column settings.
Add Custom Column Adds a custom column.
Delete Custom Deletes a custom column.
Column
Fill Down Copies the same data into the selected cells.
AutoIncrement Automatically increments data into the selected cells.
Delete Samples Deletes the selected row.
Select Autosampler Selects an autosampler.

Queue States and Device Status

The Queue Manager shows queue, batch, and sample status. Detailed information about a particular sample
in the queue can also be viewed.

Tip! Click to view the queue.

Queue States
The current state of the queue is indicated in the Queue Server.

Figure 12-7 Queue Server Indicator Showing Normal Mode

Figure 12-8 Queue Server Indicator Showing Tune Mode

The first icon indicates the queue state. The second icon indicates whether the queue is in Tune mode (for tuning)
or Normal mode (for running samples). Table 12-1 describes the icons and queue states.

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Table 12-1 Queue States

Icons State Definition

Not Ready The hardware profile is deactivated and the queue is not
accepting any sample submissions.

Stand By The hardware profile has been activated, but all devices are
idle. Pumps are not running and gases are turned off.

Warming Up The mass spectrometer and devices are equilibrating, columns

are being conditioned, the autosampler needle is being washed,
and column ovens are reaching temperature. The duration of
equilibration is selected by the operator. From this state, the
system can go to the Ready state.
Ready The system is ready to start running samples and the devices
have been equilibrated and are ready to run. In this state, the
queue can receive samples and will run after samples are
submitted.
Waiting The system will automatically begin acquisition when the next
sample is submitted.

PreRun The method is being downloaded to each device and device

equilibration is occurring. This state occurs before the acquisition
of each sample in a batch.

Acquiring The method is running and data acquisition is occuring.

Paused The system has been paused during acquisition.

View Instrument and Device Status Icons

Icons representing the mass spectrometer and each device in the active hardware configuration are shown on the
status bar in the bottom right corner of the window. The user can view the detailed status of an LC pump to

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Operating Instructions — Batches

determine whether the LC pump pressure is appropriate or view the detailed status of the mass spectrometer to
confirm the temperature of the ion source.

Note: For each status, the background color can be red. A red background indicates that the device encountered
an error while in that state.

• On the status bar, double-click the icon for the device or mass spectrometer.

The Instrument Status dialog opens.

Table 12-2 Instrument and Device Status Icons

Status Icon Background Description

Color
Idle Green or yellow The device is not running. If the background color is
yellow, then the device should be equilibrated before
it is ready to run. If the background color is green, the
device is ready to run.
Equilibrating Green or yellow The device is equilibrating.

Waiting Green The device is waiting for a command from the software
or another device, or for some action by the operator.
Running Green The device is running a batch.

Aborting Green The device is aborting a run.

Downloading Green A method is being transferred to the device.

Ready Green The device is not running, but is ready to run.

Error Red The device has encountered an error that should be

investigated.

Queue Right-click Menu

Right-click in the Queue table to access the options.

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Figure 12-9 Queue Manager Right-Click Menu

Menu Function
Sample Details Opens the Sample Details dialog.
Reacquire Acquires a sample again.
Insert Pause Inserts a pause, in seconds, between two samples.
Delete Deletes either the batch or the selected samples.
Move Batch Moves the batch within the queue.
Sort Sorts on the preselected column.
Column Settings Changes the column settings.

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Service and Maintenance
Information 13
Regularly clean and maintain the system for optimal performance.

WARNING! Electrical Shock Hazard. Do not remove the covers. Removing the
covers might cause injury or malfunctioning of the system. The covers need
not be removed for routine maintenance, inspection, or adjustment. Contact
a SCIEX Field Service Employee (FSE) for repairs that require the covers to be
removed.

WARNING! Radiation Hazard, Biohazard, or Toxic Chemical Hazard. Determine

whether decontamination is required prior to cleaning or maintenance. The
customer must decontaminate the system prior to cleaning or maintenance
if radioactive materials, biological agents, or toxic chemicals have been used
with the system.

Recommended Maintenance Schedule

Table 13-1 provides a recommended schedule for cleaning and maintaining the system.

Tip! Perform maintenance tasks regularly to make sure that the mass spectrometer is performing optimally.

For information on maintaining the ion source, refer to the ion source Operator Guide.
Contact a Qualified Maintenance Person (QMP) to order consumable parts. Contact a SCIEX Field Service Employee
(FSE) for maintenance service and support.

Table 13-1 Maintenance Tasks

Component Frequency Task For more information...

System
Tubing Daily Inspect the tubing Refer to Chemical Precautions on page
and fittings to make 11.
sure that they are
securely connected,
and that there are
no leaks.

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 Service and Maintenance Information

Table 13-1 Maintenance Tasks (continued)

Component Frequency Task For more information...

Mass Spectrometer
Curtain plate Daily Clean Refer to Clean the Curtain Plate on
page 156.
Orifice plate (front) Daily Clean Refer to Clean the Front of the Orifice
Plate on page 157.
Roughing pump oil Weekly Inspect the level Refer to Inspect the Roughing Pump Oil
Level on page 158.
Mass spectrometer Every 6 months Replace Contact the local QMP or FSE.
air filter
Roughing pump oil Annually Replace Contact the local QMP or FSE.
Instrument As needed Clean Refer to Clean the Surfaces on page
surfaces 152.
Source exhaust As needed Empty Refer to Empty the Source Exhaust
drain bottle Drain Bottle on page 158.
Orifice plate (front As needed Clean Contact the local QMP or FSE.
and rear)
®
QJet ion guide As needed Clean Contact the local QMP or FSE.
and IQ0 lens
Q0 rod set and IQ1 As needed Clean Contact the local QMP or FSE.
lens
Roughing pump oil As needed Refill Contact the local QMP or FSE.
Interface heater As needed Replace Contact the local QMP or FSE.
Ion Source
®
TurboIonSpray As needed Inspect and replace Refer to the ion source Operator
and APCI Guide.
electrodes
Corona discharge As needed Replace Refer to the ion source Operator
needle Guide.
Turbo heater As needed Replace Contact the local QMP or FSE.
Sample tubing As needed Replace Refer to the ion source Operator
Guide.

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Service and Maintenance Information

For “As needed” tasks, follow these guidelines:

• Clean the mass spectrometer surfaces after a spill or when they become dirty.
• Empty the drain bottle before it becomes full.
®
• Clean the orifice plate, QJet ion guide, and Q0 region if system sensitivity degrades.

Tip! Clean the Q0 region regularly to minimize the impact of charging (a significant loss of sensitivity of the
ions of interest over a short period of time) on the quadrupoles and lenses. Contact a QMP or FSE.

®
• Clean the QJet ion guide and Q0 region if system sensitivity degrades.

Tip! Clean the Q0 region regularly to minimize the impact of charging (a significant loss of sensitivity of the
ions of interest over a short period of time) on the quadrupoles and lenses. Contact a QMP or FSE.

• Refill the roughing pump oil when it falls below the minimum oil level.

Clean the Surfaces

Clean the external surfaces of the mass spectrometer after a spill or when they become dirty.

CAUTION: Potential System Damage. Use only the recommended cleaning methods and
materials to avoid damaging the equipment.

1. Wipe the external surfaces with a soft cloth dampened with warm, soapy water.
2. Wipe the external surfaces with a soft cloth moistened with water to remove any soap residue.

Clean the Front-End

The following warning applies to all of the procedures in this section:

WARNING! Hot Surface Hazard. Let the ion source cool for at least 30 minutes
before starting any maintenance procedures. Surfaces of the ion source and
the vacuum interface components become hot during operation.

Clean the mass spectrometer front-end using the routine cleaning method, to:
• Minimize unscheduled system downtime.
• Maintain optimum sensitivity.
• Avoid more extensive cleaning that requires a service visit.

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 Service and Maintenance Information

When contamination occurs, perform an initial routine cleaning. Clean up to and including the front of the orifice
plate. If routine cleaning does not resolve issues with sensitivity, then a full cleaning might be necessary. Contact
the local QMP or FSE.
This section provides instructions for performing routine cleaning without breaking vacuum.

Note: Follow all of the applicable local regulations. For health and safety guidelines, refer to Chemical Precautions
on page 11.

Symptoms of Contamination
The system might be contaminated if any one of the following is observed:
• Significant loss in sensitivity
• Increased background noise
• Additional peaks that are not part of the sample are shown in full scan or survey scan methods
If any of these issues are observed, then clean the mass spectrometer front-end.

Required Materials

Note: U.S. customers can call 877-740-2129 for ordering information and inquiries. International customers
can visit sciex.com/contact-us.

• Powder-free gloves (nitrile or neoprene recommended)

• Safety glasses
• Laboratory coat
• Fresh, high-quality (pure) water (at least 18 MΩ de-ionized [DI] water or ultra-pure HPLC-grade water). Old
water can contain contaminants that can further contaminate the mass spectrometer.
• MS-grade methanol, isopropanol (2-propanol), or acetonitrile
• Cleaning solution. Use one of:
• 100% methanol
• 100% isopropanol
• 1:1 acetonitrile:water solution (freshly prepared)
• 1:1 acetonitrile:water with 0.1% acetic acid solution (freshly prepared)
• Clean 1 L or 500 mL glass beaker to prepare cleaning solutions
• 1 L beaker to catch used solvent
• Organic waste container

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Service and Maintenance Information

• Lint-free wipes. Refer to Tools and Supplies Available from the Manufacturer on page 154.
• (Optional) Polyester (poly) swabs

Tools and Supplies Available from the Manufacturer

Description Part Number

Small poly swab (thermally bonded). Also available in the Cleaning kit. 1017396
Lint-free wipe (11 cm x 21 cm, 4.3 inches x 8.3 inches). Also available in the Cleaning 018027
kit.
Cleaning kit. Contains the small poly swab, lint-free wipes, Q0 cleaning tool, straight 5020761
®
QJet ion guide cleaning brush, and Alconox packets.

Cleaning Best Practices

WARNING! Hot Surface Hazard. Let the ion source cool for at least 30 minutes
before starting any maintenance procedures. Surfaces of the ion source and
the vacuum interface components become hot during operation.

WARNING! Toxic Chemical Hazard. Refer to the chemical product Safety Data Sheets
and follow all of the recommended safety procedures when handling, storing, and
disposing of chemicals. For health and safety precautions, refer to the System User
Guide.

WARNING! Radiation Hazard, Biohazard, or Toxic Chemical Hazard. Determine

whether decontamination is required prior to cleaning or maintenance. The
customer must decontaminate the system prior to cleaning or maintenance
if radioactive materials, biological agents, or toxic chemicals have been used
with the system.

WARNING! Environmental Hazard. Do not dispose of system components in municipal

waste. Follow local regulations when disposing of components.

• Allow the ion source to cool before removing it.

• Always wear clean, powder-free gloves (nitrile or neoprene recommended) for the cleaning procedures.
• After cleaning the mass spectrometer components, and before reassembling them, put on a new, clean pair
of gloves.

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 Service and Maintenance Information

• Do not use cleaning supplies other than those specified in this procedure.
• If possible, prepare cleaning solutions just before cleaning.
• Prepare and store all of the organic solutions and organic-containing solutions in very clean glassware only.
Never use plastic bottles. Contaminants can leach from these bottles and further contaminate the mass
spectrometer.
• To avoid contaminating the cleaning solution, pour the solution on the wipe or swab.
• Allow only the center area of the wipe to contact the mass spectrometer surface. Cut edges can leave fibers
behind.

Tip! Wrap the wipe around a thermally-bonded poly swab.

Figure 13-1 Example: Folding the Wipe

• To avoid cross-contamination, discard the wipe or swab after it has touched the surface once.
• Larger parts of the vacuum interface, such as the curtain plate, might require several cleanings, using multiple
wipes.
• Only dampen the wipe or swab slightly when applying water or cleaning solution. Water, more often than
organic solvents, might cause the wipe to deteriorate, leaving residue on the mass spectrometer.
• Do not rub the wipe across the aperture. Wipe around the aperture to prevent fibers from the wipes from
entering the mass spectrometer.
• Do not insert the brush in the aperture on the curtain plate or orifice plate.

Prepare the Mass Spectrometer

®
Note: Mass spectrometers with a NanoSpray ion source might require a full cleaning for best results. Contact
a local QMP or an FSE.

WARNING! Hot Surface Hazard. Let the ion source cool for at least 30 minutes
before starting any maintenance procedures. Surfaces of the ion source and
the vacuum interface components become hot during operation.

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Service and Maintenance Information

CAUTION: Potential System Damage. Do not drop anything into the source drain when the
ion source is removed.

Figure 13-2 Source Drain on the Vacuum Interface

1. Deactivate the hardware profile.

2. Remove the ion source. Refer to the ion source Operator Guide.

When the ion source is not in use, store it to protect it from damage and to maintain operating integrity.

Clean the Curtain Plate

CAUTION: Potential System Damage. Do not rest the curtain plate or orifice plate on the
aperture tip. Make sure that the conical side of the curtain plate faces up.

CAUTION: Potential System Damage. Do not insert a wire or metal brush into the aperture
on the curtain plate, orifice plate, or interface heater to avoid damaging the aperture.

1. Pull the curtain plate off of the vacuum interface and then put it, conical side up, on a clean, stable surface.

The curtain plate is held in place by three retaining ball catches mounted on the orifice plate.

Tip! If the curtain plate does not immediately separate from the orifice plate, then turn the curtain plate
slightly (less than 90 degrees) to release the ball spring latches.

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 Service and Maintenance Information

2. Dampen a lint-free wipe with pure water and then clean both sides of the curtain plate.

Note: Use multiple wipes, as required.

3. Repeat step 2 using the cleaning solution.

4. Using a dampened wipe or small poly swab, clean the aperture.
5. Wait until the curtain plate is dry.
6. Inspect the curtain plate for solvent stains or lint, removing any residue with a clean, slightly damp, lint-free
wipe.

Note: Persistent spotting or filming is an indicator of contaminated solvent.

Clean the Front of the Orifice Plate

CAUTION: Potential System Damage. When cleaning the surface of the orifice plate, do
not remove the interface heater. Frequent removal of the interface heater can result in
damage to the interface heater. Surface cleaning of the interface heater is adequate for
routine cleaning.

CAUTION: Potential System Damage. Do not insert a wire or metal brush into the aperture
on the curtain plate, orifice plate, or interface heater to avoid damaging the aperture.

1. Dampen a lint-free wipe with water and then wipe the front of the orifice plate, including the interface heater.
2. Repeat step 1 using the cleaning solution.
3. Wait until the orifice plate is dry.
4. Inspect the orifice plate for solvent stains or lint, removing any residue with a clean, slightly damp, lint-free
wipe.

Note: Persistent spotting or filming is an indicator of contaminated solvent.

Put the Mass Spectrometer Back in Service

1. Install the curtain plate on the mass spectrometer.
2. Install the ion source on the mass spectrometer. Refer to the ion source Operator Guide.
3. Activate the hardware profile. Refer to the System User Guide.

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Service and Maintenance Information

Inspect the Roughing Pump Oil Level

• Inspect the sight glass on the roughing pump to verify that the oil is above the minimum level.

If the oil is below the minimum level, then contact the qualified maintenance person (QMP) or SCIEX field
service employee (FSE).

Empty the Source Exhaust Drain Bottle

WARNING! Radiation Hazard, Biohazard, or Toxic Chemical Hazard. Deposit

hazardous materials in appropriately labeled waste containers and dispose
of them according to local regulations.

WARNING! Radiation Hazard, Biohazard, or Toxic Chemical Hazard. Take care

to vent exhaust gases to a dedicated laboratory fume hood or exhaust system
and make sure that the ventilation tubing is secured with clamps. Make sure
that the laboratory has appropriate air exchange for the work performed.

Inspect the source exhaust drain bottle regularly, and empty it before it becomes full. Also inspect the bottle and
its fitting for leaks, and tighten connections or replace components, if required. Follow the steps in this procedure
to empty the bottle.
1. Remove the ion source. Refer to the ion source Operator Guide.
2. Loosen the clamps connecting the hoses to the cap of the source exhaust drain bottle.
3. Disconnect the hoses from the cap.
4. If applicable, lift the drain bottle out of the holder.
5. Remove the cap from the drain bottle.
6. Empty the drain bottle and then dispose of the waste according to laboratory procedures and local waste
regulations.
7. Install the cap on the bottle and then put the bottle in the holder.
8. Attach the hoses to the cap and then secure them tightly with clamps.

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Figure 13-3 Source Exhaust Drain Bottle

Item Description
1 Connection to vent
2 Source exhaust drain tubing: 2.5 cm (1.0 inch) inside diameter (i.d.)
3 Roughing pump exhaust hose: 3.2 cm (1.25 inch) i.d.
4 Source exhaust drain bottle. Make sure that the bottle is secured to prevent spillage.
5 Connection to the mass spectrometer: 1.6 cm (0.625 inch) i.d.

Note: Source exhaust hose connections at the drain bottle, mass spectrometer, and the lab vent are secured
with hose clamps.

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Service and Maintenance Information

Storage and Handling

WARNING! Environmental Hazard. Do not dispose of system components in municipal

waste. Follow local regulations when disposing of components.

If the mass spectrometer must be stored for a long time or prepared for shipping, then contact a SCIEX FSE for
decommissioning information. To disconnect power from the mass spectrometer, remove the mains supply connector
from the AC mains supply.

Note: The ion source and mass spectrometer must be transported and stored between –30 °C to +60 °C (–22
°F to 140 °F). Store the system at an altitude not exceeding 2 000 m (6 562 feet) above sea level.

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Mass Spectrometer
Troubleshooting 14
This section contains information for troubleshooting basic system issues. Certain activities can only be carried
out by a SCIEX -trained Qualified Maintenance Person (QMP) in the laboratory. For advanced troubleshooting,
contact a SCIEX Field Service Employee (FSE).

Table 14-1 System Issues

Symptom Possible Cause Corrective Action

® TM
The QJet ion guide is extremely dirty The Curtain Gas flow rate is too Verify the setting for the CUR
or frequently becomes dirty. low. parameter, and increase it, if
applicable.
A system fault has occurred because 1. The roughing pump oil level is 1. Inspect the roughing pump oil
the vacuum pressure is too high. too low. level, and then contact the local
QMP or an FSE to add oil.
2. There is a leak.
2. Inspect and repair leaks.
3. The wrong orifice plate is
installed. 3. Install the correct orifice plate.

A system fault has occurred because 1. The mass spectrometer air filter Contact the local QMP or FSE.
the QPS exciter module temperature is blocked.
is too high.
2. Ambient temperature is too high.
®
The Analyst software reports that 1. The probe is not installed. 1. Confirm the fault in the Status
the mass spectrometer is in Fault panel of the device details page.
2. The probe is not connected
status because of the ion source.
securely. 2. Install the probe. Refer to the ion
source Operator Guide.
3. Remove and replace the probe.
Tighten the retaining ring
securely. Refer to the ion source
Operator Guide.
®
The Analyst software indicates that The F3 fuse is blown. Contact an FSE.
the APCI probe is in use, but the
®
TurboIonSpray probe is installed.
The spray is not uniform. The electrode is blocked. Clean or replace the electrode. Refer
to the ion source Operator Guide.

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Mass Spectrometer Troubleshooting

Table 14-1 System Issues (continued)

Symptom Possible Cause Corrective Action

Sensitivity is reduced. 1. The ion source parameters are 1. Optimize the ion source
not optimized. parameters. Refer to
®
theAnalyst software Help
2. The mass spectrometer is not
system.
optimized.
2. Refer to Clean the Curtain Plate
3. The curtain plate is dirty.
on page 156.
4. The orifice plate is dirty.
®
3. Refer to Clean the Front of the
5. The QJet ion guide or IQ0 lens Orifice Plate on page 157 or
is dirty. contact the local QMP or FSE.
6. The Q0 region is dirty. 4. Clean the Q0 region. Contact the
7. The syringe or sample line is QMP or FSE.
leaking. 5. Inspect the syringe or sample line
8. The sample has degraded or has for leaks, and repair any leaks
a low concentration. found. Make sure that all fittings
are the correct type and size.
9. The probe is not installed
properly. 6. Verify the sample concentration.
Use a fresh sample.
10. The ion source is not installed
properly, or it is faulty. 7. Remove and install the probe.

11. One or more of the O-rings on the 8. Remove and install the ion
vacuum interface is missing. source, making sure that the
latches are properly secured. If
12. There is an issue with the LC this does not resolve the issue,
system or connections. then install and optimize an
alternate ion source.
9. If the O-rings are on the ion
source, then install them on the
vacuum interface. If they are
missing, then contact an FSE.
10. Troubleshoot the LC system.

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 Mass Spectrometer Troubleshooting

Table 14-1 System Issues (continued)

Symptom Possible Cause Corrective Action

The mass spectrometer performance 1. The probe is not optimized. 1. Optimize the probe. Refer to the
has degraded. ion source Operator Guide.
2. The sample was not prepared
correctly or the sample has 2. Confirm that the sample was
degraded. prepared correctly.
3. The sample inlet fittings are 3. Verify that the fittings are the
leaking. right size and type and make sure
that they are tight. Do not
overtighten the fittings. Replace
the fittings if leaks continue.
4. Install and optimize an alternate
ion source.
5. Contact an FSE if the issue
persists.

Arcing or sparks occur. The position of the corona discharge Turn the corona discharge needle
needle is incorrect. toward the curtain plate, and away
from the stream of heater gas. Refer
to the ion source Operator Guide.

For sales, technical assistance, or service, contact an FSE or visit the SCIEX Web site at sciex.com for contact
information.

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Operating Instructions — Manual
Compound Optimization A
The user must control the autosampler and injection valve manually, as these devices cannot be controlled through
the system while it is in Tune and Calibrate mode.

Prerequisites
• The mass spectrometer is tuned and calibrated.
• Conditions for an LC separation are known.
• All of the required peripheral devices, including the syringe pump, if needed, and LC components are in the
hardware profile.

Required Materials
To tune instrument parameters for particular compounds the following solutions are recommended. The mixture
of four compounds is used for illustrating the steps of the procedure.
• Mobile phase: 1:1 acetonitrile:water + 2 mM ammonium acetate + 0.1% formic acid.
• LC pump and autosampler.
• Autosampler vials.

Table A-1 Compounds and Molecular Weights

Compound m/z
Minoxidil 210.2
Tolbutamide 271.1
Reserpine 609.3
Rescinnamine 635.3

About Manual Compound Optimization

Manual compound optimization is used to optimize compound- and ion source-dependent parameters for an
analyte. When the user manually optimizes for an analyte, an MS acquisition method is created in Tune and

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 Operating Instructions — Manual Compound Optimization

Calibrate mode. Depending on the method of sample introduction selected, add an LC method to the acquisition
method so that infusion or LC can be used.
Optimizing to give the highest signal does not always give the highest signal-to-noise ratios. Noise can scale with
signal for some parameters and should be verified during optimization if the goal is to obtain maximum
signal-to-noise ratio.
When optimizing ion source-dependent parameters, introduce the sample at flow rates that will be used during
sample analysis, using either flow injection analysis (FIA) or Tee-infusion as the method of sample introduction.
The CAD gas is the only compound-dependent parameter that is shown on the Source/Gas tab and can be easily
optimized while infusing the analyte.
Optimize the position of the ion source before optimizing the ion source-dependent parameters. Refer to the ion
source Operator Guide.

About Scan Types

For this example, use the Q1 MS, Q1 MI, Product Ion, and MRM scan types. The Q1 MS scan type is used to confirm
the presence of compounds of interest. The Q1 MI scan is used to optimize MS or pre-collision cell voltages. The
Product Ion scan type is used to determine the product ions of each compound. The MRM scan type is used to
optimize the collision energy (CE) and collision cell exit potential (CXP) for each product ion or fragment. Use the
methods created in this section for quantitative or qualitative analysis.

Manually Optimize an Analyte

After the acquisition method is created, optimize compound-dependent parameters using the Edit Ramp function
or by manually editing the parameters in the Tune Method Editor. Ion source-dependent parameters can be
optimized only by manually adjusting the parameters in the Tune Method Editor. Depending on the scan type
used, different parameters are available to optimize.
Follow the procedures in the order given:
1. Confirm the Presence of Compounds on page 165
2. Optimize MS-Specific Parameters on page 167
3. Determine the Product Ions for Optimization on page 168
4. Determine the Product Ions for Optimization on page 168
5. Optimize Collision Cell Exit Potential for each Product Ion on page 170

Confirm the Presence of Compounds

1. Create a project.
2. Activate the hardware profile.

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Operating Instructions — Manual Compound Optimization

3. Prepare the sample:

a. Aspirate the compound solution into a syringe and remove the air from the syringe.
b. Use the tubing with the special fitting to connect the syringe to the mass spectrometer.
c. Install the syringe in the integrated syringe pump.
4. Infuse the compound in solution at a rate of 5 µL/min to 10 µL/min.
5. On the Navigation bar, under Tune and Calibrate, double-click Manual Tuning.
6. On the Syringe Pump Method Properties tab, type the parameter values shown in Table A-2.

Table A-2 Syringe Pump Method Properties Tab

Parameter Value
Syringe Diameter Syringe dependent. 4.610 mm if a 1.0 mL syringe is used
Flow Rate 10
Unit µL/min

Figure A-1 Syringe Pump Method Properties Tab

7. Click Start Syringe Pump.

8. Click MS Method from the method list.
9. Click Start.
10. Wait until an even TIC is shown on the left and peaks are shown on the right and then click Stop.
11. Select the MCA check box.
12. Type 10 In the Cycles field.
13. Click Start.
14. When ten scans are complete, the masses of the four compounds are shown as ions.

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 Operating Instructions — Manual Compound Optimization

Tip! If the previous or expected ion chromatogram pane is hidden, then select the window to be shown
from the Window menu.

Note: Ion intensities of the compounds can show large variances. To facilitate moving to a solution of higher
or lower concentration as needed during the optimization, have several concentration levels prepared before
beginning optimization.

15. Right-click the bottom-right spectral pane and then click Open File.
16. Find the compounds of interest and then write down the m/z values for the highest peaks. These values should
be within 0.1 Da to 0.2 Da of the expected m/z. In the next procedure, use the m/z values.

Optimize MS-Specific Parameters

Declustering potential (DP) and entrance potential (EP) are pre-collision cell voltages. Optimization of these values
involves gradually changing the voltage range while monitoring the signal intensity of the compound.
The DP is the difference between the orifice and the ground. The higher the potential difference, the greater the
amount of declustering.
The DP parameter has a significant effect on analyte signal. Typical DP values range from 20 V to 150 V. A DP
value that is too low will result in lower ion intensity and potential interferences from clusters. A DP value that is
too high can cause fragmentation of the analyte in the source. Generally, the DP should be set to the value that
provides the highest intensity.
The EP parameter controls the entrance potential, which guides and focuses the ions through the high-pressure
Q0 region. It is typically set at 10 V for positive ions or –10 V for negative ions. The EP has a minor effect on
compound optimization and can generally be left at default values without impact on analyte detection limits.
1. Return to the Tune Method Editor and change the method to the Q1 Multiple Ions (Q1 MI) scan type.
2. In the mass table, type the appropriate parameter values. Refer to Table A-3.

Table A-3 Mass Table Parameters—Q1 Multiple Ions (Q1 MI)

Compound Q1 Mass Time

Reserpine 609.4 1
Minoxidil 210.2 1
Tolbutamide 271.3 1
Rescinnamine 635.4 1

Start with reserpine for a simple case. Repeat the manual optimization process for the remaining compounds.
3. Click Edit Ramp.

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Operating Instructions — Manual Compound Optimization

4. Select Declustering Potential (DP) in the Ramp Parameter Settings dialog.

Note: Start with the DP parameter and then optimize the other parameters in the order that they are shown
in the dialog. The parameters might not be optimized correctly if they are optimized out of order.

5. Type the required Start, Stop, and Step values.

Tip! The existing values are good starting points. Use the Edit Ramp function to change these values to
be more efficient.

6. Click OK.
7. Click Start.
8. Right-click the bottom-right XIC pane and then click Open File to maximize the XIC view.
9. Monitor the XICs.

Note: The value that gives the best signal per second for the ion of interest is the optimal value.

10. Note the optimal value for the ion of interest.

11. Move the cursor to the mass table, right-click, and then add the parameter just optimized.

A column is added to the table.

12. Add the optimized value to the appropriate row.
13. Repeat these steps for each mass in the acquisition method until there is a list of optimal values for all of the
masses.
14. Repeat these steps to optimize for the other MS-specific parameters.

Table A-4 MS-Specific Parameters

Parameter Comment
DP Set the DP to the value that provides the highest intensity.
EP Rarely optimize this parameter because it has a smaller effect.

Determine the Product Ions for Optimization

The collision energy (CE) controls the amount of energy that the precursor ions receive as they are accelerated
into the collision cell.

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 Operating Instructions — Manual Compound Optimization

Perform this procedure, one compound at a time, using the MS-specific optimized values that were obtained
previously. The product ions provide the Q3 mass of MRM transitions.
In this example, the compound reserpine is used.
1. In the Tune Method Editor, close the XIC panes.
2. Click Product Ion (MS2) in the Scan type field.
3. Click the Compound tab and then type the optimal value noted previously.
4. On the MS tab, in the Product Of field, type 609.4. This value is the mass assignment for reserpine that
was noted in Confirm the Presence of Compounds on page 165.
5. Make sure that the Center / Width check box is not selected.
6. In the mass table, type the appropriate Start, Stop, and TIme values. Refer to Table A-5.

Table A-5 Mass Table Parameters (Product Ion Scan)

Field Value
Start (Da) 100
Stop (Da) 650
Time (sec) 2

7. Click Edit Ramp.

8. In the Ramp Parameter Settings dialog, select Collision Energy and then type the required Start, Stop,
and Step values.

Note: The existing values are good starting points. Use the Edit Ramp function to change these values
to be more efficient.

9. Click OK.
10. Select the MCA check box.
11. Click Start.
12. Right-click the bottom-right XIC pane and then click Open File.
13. Select the product ions with the greatest intensity and note the product ion m/z value to the first decimal
point, such as 195.1.

We recommend that two or three product ions be optimized for each compound. The additional transitions
can be used for confirmation, or to avoid the need to re-optimize a compound in case an interference is found.

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Operating Instructions — Manual Compound Optimization

Note: Make sure that the highest peaks selected for optimization do not represent a common loss from the
precursor ion, such as water or carbon dioxide. Also make sure that the product ion is not too low in mass
or interferences might occur for that transition in real samples or clusters from the mobile phase when
analyzing on column.

14. Repeat this procedure for the remaining compounds.

Optimize Collision Cell Exit Potential for each Product Ion

1. In the Tune Method Editor, close the XIC panes.
2. Open the previously saved method.
3. In the mass table, verify the Q1 and Q3 m/z values for the compound.
4. Click the Compound tab and then type the optimal DP and CE values noted previously.
5. Click Edit Ramp.
6. Select Collision Cell Exit Potential (CXP) in the Ramp Parameter Settings dialog.
7. Type the required Start, Stop, and Step values.

Tip! The existing values are good starting points. Use the Edit Ramp function to change these values to
be more efficient.

8. Click OK.
9. Click Start.
10. Right-click the bottom-right XIC pane and then click Open File.
11. Note the optimal value for the ion of interest.

The value that gives the best signal is the optimal value.
12. In the mass table, right-click and then select the parameter just optimized.

This adds a column to the table.

13. Repeat if other product ions were monitored.
14. Add the optimized values to the appropriate row.
15. Save the method.
16. Repeat this procedure for any other compounds that were previously optimized.

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 Operating Instructions — Manual Compound Optimization

Manually Optimize the Ion Source and Gas

Parameters
The ion source and gas settings must be set correctly to prevent contamination of the mass spectrometer and to
make sure that the compounds of interest are optimally put into the gas phase as ions.

Note: The optimal ion source and gas settings are related to the solvent composition and the flow rate.

The ion source and gas settings must be adjusted when the LC conditions change significantly.
To optimize the ion source and gas parameters, set up a syringe pump with the compounds of interest and connect
the line with a tee to the LC device. Controlling the LC pump can be done manually or through the software.
Another way to manually optimize ion source and gas settings is to use the autosampler to manually inject the
compound of interest while manually varying the parameters in manual tuning to find the optimal settings.

Prepare the Ion Source

1. Set the horizontal micrometer to 5.
2. Set the vertical micrometer on the ion source for the flow rate.

Use the parameters in Table A-6.

TM
Table A-6 Turbo V Ion Source Vertical Parameters

Flow Rate Initial Vertical Parameters

1 µL/min to 20 µL/min 10
20 µL/min to 250 µL/min 5
250 µL/min to 500 µL/min 2
500 + µL/min 0

Depending on how the optimization is run, a hardware profile with the LC pumps might need to be configured.
Refer to the ion source Operator Guide.

Optimize the Ion Source Parameters

TM
Ion source parameters are optimized for best signal-to-noise for the compound of interest. The Curtain Gas
supply is optimized at the highest setting without losing sensitivity. Refer to the ion source Operator Guide.
TM TM
Use the following procedure to optimize the Curtain Gas flow parameter. The main function of the Curtain Gas
TM
flow parameter is to prevent the contamination of the ion optics. The Curtain Gas flow parameter should always

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Operating Instructions — Manual Compound Optimization

be maintained as high as possible without losing sensitivity. The value depends on the type of mass spectrometer
and ion source.
Do not set the parameter below the starting value.
The minimum flow rate supported by the APCI probe is 200 µL/min.

Note: Depending on the mass spectrometer, all parameters might not be available.

1. On the Navigation bar, under Tune and Calibrate, double-click Manual Tuning.
2. Click File > Open.
3. In the Files list, click the acquisition method used to optimize the compound parameter and then click OK.

The method opens in the Tune Method Editor.

4. Click the Source/Gas tab.
5. Using the ion source and gas flow guide, set all of the ion source and gas parameters so that they are appropriate
for the flow rate.
6. Set the run time long enough so that many parameters can be adjusted. A recommended starting time is 15
minutes.
7. Click Start.

Data is shown in panes below the Tune Method Editor.

8. Note the signal of the peak of interest.
9. In the Curtain Gas (CUR) field, increase the value by five.
10. Continue increasing the Curtain Gas (CUR) value until the highest value without losing sensitivity is found.

As with most Source/Gas parameters, if two values give the same result, then use the higher value.
11. Repeat this procedure for the other Source/Gas parameters.

When optimizing for these parameters, look for the value that gives the highest signal-to-noise value.

Advanced Parameters
The following parameters should only be optimized by an experienced operator.

Optimize AF2
The AF2 parameter controls the fragmentation of the second precursor ion in an MS3 scan. The amount of excitation
energy used depends on the compound and the desired amount of fragmentation.

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 Operating Instructions — Manual Compound Optimization

1. For both positive and negative ion modes, ramp AF2 from 0.07 V to 0.3 V, with a step value of 0.01 V.

The allowable range is 0 V to 1 V.

2. For both positive and negative ion modes, ramp AF2 from 0 mV to 200 mV with a step value of 5 mV.
3. Select the AF2 value that gives the best amount of fragmentation.

+
If the MS3 scan is selected when using the 6500 and 6500 series of instruments, the No Fragmentation
option is not available. However, users can achieve the effect of the No Fragmentation option by setting the
AF2 parameter to 0 on the Compound tab of the Tune Method Editor.

About Collision Energy Spread (CES)

Under the compound-dependent parameters in Enhanced Product Ion (EPI) and MS3 experiments, the collision
energy spread (CES) field allows users to specify the difference in collision energies that will be applied in the
experiment. For example, if CE value of 30 and a CES value of 15 are used, then collision energies of 15, 30, and
45 are used.

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5500 Series System Parameters B
The first number under each scan type is the preset value. The range of numbers is the accessible range for each
parameter.

Table B-1 5500 Series System Parameters

Parameter Access ID Positive Ion Mode Negative Ion Mode

ID
Q1 Q3 MS/MS Q1 Q3 MS/MS
CUR CUR 20 20 20 20 20 20
10 to 55 10 to 55 10 to 55 10 to 55 10 to 55 10 to 55
CAD CAD 0 6 Med (9) 0 5 Med (9
n/a n/a 0 to 12 n/a n/a 0 to 12
(1)(2) (1)(2)
IS IS 5500 5500 5500 –4500 –4500 –4500
0 to 5500 0 to 5500 0 to 5500 –4500 to 0 –4500 to 0 –4500 to 0
(3) (3)
NC NC 3 3 3 –3 –3 –3
0 to 5 0 to 5 0 to 5 –5 to 0 –5 to 0 –5 to 0
(2)(3) (2)(3)
TEM TEM 0 0 0 0 0 0
0 to 750 0 to 750 0 to 750 0 to 750 0 to 750 0 to 750
OR DP 100 100 100 –100 –100 –100
(DP = OR) 0 to 300 0 to 300 0 to 300 –300 to 0 –300 to 0 –300 to 0
Q0 EP 10 10 10 –10 –10 –10
(EP = –Q0) 2 to 15 2 to 15 2 to 15 –15 to –2 –15 to –2 –15 to –2
IQ1 IQ1 Q0 + (–0.5) Q0 + Q0 + Q0 + 0.5 Q0 + 0.5 Q0 + 0.5
(–0.5) (–0.5)
(IQ1 = Q0 + –0.1 to –2 0.1 to 2 0.1 to 2 0.1 to 2
offset) –0.1 to –2 –0.1 to –2
ST ST Q0 + (-8) Q0 + (-8) Q0 + (-8) Q0 + 8 Q0 + 8 Q0 + 8
(ST = Q0 + –12 to –5 –12 to –5 -12 to –5 12 to 5 12 to 5 12 to 5
offset)

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 5500 Series System Parameters

Table B-1 5500 Series System Parameters (continued)

Parameter Access ID Positive Ion Mode Negative Ion Mode

ID
Q1 Q3 MS/MS Q1 Q3 MS/MS
RO1 IE1 1 n/a 1 –1 n/a –1
(IE1 = Q0 – 0 to 3 0 to 3 –3 to –0 –3 to –0
RO1)
IQ2 IQ2 Q0+ (–10) Q0+ (–10) Q0+ (–10) Q0 + 10 Q0 + 10 Q0 + 10
(IQ2 = Q0 +
–30 to –8 –30 to –8 –30 to –8 8 to 30 8 to 30 8 to 30
offset)
RO2 RO2 –20 –20 n/a 20 20 n/a
n/a n/a n/a n/a
RO2 CE n/a n/a 30 n/a n/a –30
(CE = Q0 – 5 to 180 –180 to
RO2)
–5
ST3 ST3 RO2 – 10 n/a n/a RO2 + 10 n/a n/a
(ST3 = RO2 + –30 to –5 5 to 30
offset)
ST3 CXP n/a 15 15 n/a –15 –15
(CXP = RO2 – 0 to 55 0 to 55 –55 to 0 –55 to 0
ST3)
RO3 RO3 –50 n/a n/a 50 n/a n/a
n/a n/a
RO3 IE3 n/a 1 1 n/a –1 –1
(IE3 = RO2 – 0 to 5 0 to 5 –5 to 0 –5 to 0
RO3)
DF DF –200 –200 –200 200 200 200
–300 to 0 –300 to 0 –300 to 0 0 to 300 0 to 300 0 to 300
CEM CEM 1800 1800 1800 1800 1800 1800
0 to 3300 0 to 3300 0 to 3300 0 to 3300 0 to 3300 0 to 3300
GS1 GS1 20 20 20 20 20 20
0 to 90 0 to 90 0 to 90 0 to 90 0 to 90 0 to 90

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5500 Series System Parameters

Table B-1 5500 Series System Parameters (continued)

Parameter Access ID Positive Ion Mode Negative Ion Mode

ID
Q1 Q3 MS/MS Q1 Q3 MS/MS
GS2 GS2 0 0 0 0 0 0
0 to 90 0 to 90 0 to 90 0 to 90 0 to 90 0 to 90
IHT IHT 150 150 150 150 150 150
0 to 250 0 to 250 0 to 250 0 to 250 0 to 250 0 to 250
sdp sdp 1 1 1 1 1 1
1 or 2 1 or 2 1 or 2 1 or 2 1 or 2 1 or 2
TM ® TM
(1) Turbo V ion source (2) TurboIonSpray probe (3) APCI probe (4) DuoSpray ion source (1=TurboIonSpray
probe and 2=APCI probe)

®
Table B-2 QTRAP 5500 System Parameters for LIT Scan Types Only

Parameter ID Access ID Positive Ion Mode Negative Ion Mode

CAD CAD High High
Low–High Low–High
AF2** AF2 0.100 0.100
0 or 1 0 or 1
AF3 AF3 Mass-Speed Dependent Mass-Speed Dependent
0 to 10 0 to 10
EXB EXB Mass-Speed Dependent Mass-Speed Dependent
–165 to 0 0 to 165
CES CES 0 0
0 to 50 0 to 50
ROS CE 10 –10
(Q0 - ROS) 5 to 180 –5 to –180
** MS/MS/MS only

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Calibration Ions and Solutions C

Table C-1 Tuning Frequency

Calibration Resolution Optimization

Scan Type Frequency Manual/Automated Frequency Manual/
Automated
Q1 and Q3 3 months to 6 Both 3 months to 6 Both
months months
LIT Every 2 weeks, as Both 3 months to 6 Automated only
required months

Table C-2 Suggested Tuning Solutions for the 5500 Series of Instruments

System Q1 and Q3 LIT

Positive Negative Positive and Negative
TM
SCIEX Triple Quad 5500 POS PPG, 2e-7 M NEG PPG, 3e-5 M N/A
LC-MS/MS system
®
QTRAP 5500 LC-MS/MS POS PPG, 2e-7 M NEG PPG, 3e-5 M Agilent ESI Tuning Mix
system

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Calibration Ions and Solutions

Table C-3 Q1 and Q3 PPG Ion Scans

Polarity Masses
Positive 59.05 175.13 616.46 906.67 1080.80 1196.88
Negative 45.00 585.39 933.64 1223.85 1572.10 N/A

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 Calibration Ions and Solutions

®
Table C-4 LIT Scans for the QTRAP 5500 LC-MS/MS System (Agilent)

Polarity Masses
Positive 118.09 322.05 622.03 922.01
Negative 112.99 431.98 601.98 N/A

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Toolbar Icons D
For additional toolbar icons, refer to the Advanced User Guide.

Table D-1 Tool Bar Icons

Icon Name Description

New Subproject Creates a subproject. Subprojects can only be created later in the process
if the project was originally created with subprojects.
Copy Subproject Copies a Subproject folder.
Subprojects can be copied only from another project that has existing
subprojects. If the same folders exist at both the project and subproject levels,
then the software uses the project level folders.

Table D-2 Acquisition Method Editor Icons

Icon Name Description

Mass Spec Click to show the MS tab in the Acquisition Method editor.

Period Right-click to add an experiment, add an IDA Criteria Level, or delete the
period.
Autosampler Click to open the Autosampler Properties tab.

Syringe Pump Click to open the Syringe Pump Properties tab.

Column Oven Click to open the Column Oven Properties tab.

Valve Click to open the Valve Properties tab.

DAD Click to open the DAD Method Editor. Refer to Generate DAD Data on page
123.
ADC Click to open the ADC Properties tab. Refer to Show ADC Data on page 114.

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 Toolbar Icons

Table D-3 Acquire Mode Icons

Icon Name Description

View Queue Shows the sample queue.

Instrument Queue Shows a remote instrument station.

Status for Remote Shows the status of a remote instrument.

Instrument
Start Sample Starts the sample in the queue.

Stop Sample Stops the sample in the queue.

Abort Sample Aborts a sample acquisition in the middle of the processing of that sample.

Stop Queue Stops the queue before it has completed processing all the samples.

Pause Sample Inserts a pause in the queue.

Now
Insert Pause Inserts a pause before a specific sample.
before Selected
Sample(s)
Continue Sample Continues acquiring the sample.

Next Period Starts a new period.

Extend Period Extends the current period.

Next Sample Stops acquiring the current sample and starts acquiring the next sample.

Equilibrate Selects the method to be used to equilibrate the devices. This method should
be the same as the method used with the first sample in the queue.
Standby Puts the instrument in Standby mode.

Ready Puts the instrument in Ready mode.

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Toolbar Icons

Table D-3 Acquire Mode Icons (continued)

Icon Name Description

Reserve Reserves the mass spectrometer for tuning and calibrating.
Instrument for
Tuning
IDA Method Starts the IDA Method Wizard.
Wizard
Purge Modifier Starts the modifier purge from the modifier pump.

Table D-4 Tune and Calibrate Mode Icons

Icon Name Description

Calibrate from Opens the Mass Calibration Option dialog and uses the active spectrum to
spectrum calibrate the mass spectrometer.
Manual Tune Opens the Manual Tune Editor.

Compound Optimizes for a compound using infusion by FIA.

Optimization
Instrument Verifies the instrument performance, adjusts the mass calibration, or adjusts
Optimization mass spectrometer settings.
View Queue Views the sample queue.

Instrument Queue Views a remote instrument.

Status for Remote Views the status of a remote instrument.

Instrument
Reserve Reserves the instrument for tuning and calibrating.
Instrument for
Tuning
IDA Method Starts the IDA Method Wizard.
Wizard

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 Toolbar Icons

Table D-5 Explore Quick Reference: Chromatograms and Spectrum

Icon Name Description

Open Data File Opens files.

Show Next Sample Goes to the next sample.

Show Previous Goes to the previous sample.

Sample
Go To Sample Opens the Select Sample dialog.

List Data Views the data in tables.

Show TIC Generates a TIC from a spectrum.

Extract Using Extracts ions by selecting masses.

Dialog
Show Base Peak Generates a BPC.
Chromatogram
Show Spectrum Generates a spectrum from a TIC.

Copy Graph to Copies the active graph to a new window.

new Window
Baseline Subtract Opens the Baseline Subtract dialog.

Threshold Adjusts the threshold.

Noise Filter Shows the Noise Filter Options dialog, which can be used define the minimum
width of a peak. Signals below this minimum width are regarded as noise.
Show ADC Shows ADC data.

Show File Info Shows the experimental conditions used to collect the data.

Add arrows Adds arrows to the X-axis of the active graph.

Remove all arrows Removes arrows from the X-axis of the active graph.

Offset Graph Compensates for slight differences in the time during which the ADC data and
the mass spectrometer data were recorded. This is useful when overlaying
graphs for comparison.

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Toolbar Icons

Table D-5 Explore Quick Reference: Chromatograms and Spectrum (continued)

Icon Name Description

Force Peak Labels Labels all of the peaks.

Expand Selection Sets the expansion factor for a portion of a graph to be viewed in greater
By detail.
Clear ranges Returns the expanded selection to normal view.

Set Selection Defines start and stop points for a selection. This feature provides more accurate
selection than is possible by selecting the region using the cursor.
Normalize To Max Scales a graph to maximum size, so that the most intense peak is scaled to
full scale, whether or not it is visible.
Show History Shows a summary of data processing operations performed on a particular
file, such as smoothing, subtraction, calibration, and noise filtering.
Open Compound Opens the compound database.
Database
Set Threshold Adjusts the threshold.

Show Contour Plot Shows selected data as either a spectrum graph or an XIC. Additionally, for
data acquired by a DAD, a contour plot can show selected data as either a
DAD spectrum or an XWC.
Show DAD TWC Generates a TWC of the DAD spectrum.

Show DAD Generates a DAD spectrum.

Spectrum
Extract Extracts up to three wavelength ranges from a DAD spectrum to view the XWC.
Wavelength

Table D-6 Explore Toolbar Quick Reference: Overlaying Graphs

Icon Name Description

Home Graph Click to return the graph to the original scale.

Overlay Click to overlay graphs.

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 Toolbar Icons

Table D-6 Explore Toolbar Quick Reference: Overlaying Graphs (continued)

Icon Name Description

Cycle Overlays Click to cycle between overlaid graphs.

Sum Overlays Click to add the graphs together.

Table D-7 Explore Toolbar Quick Reference: Fragment Interpretation Tool

Icon Name Description

Show Fragment Click to open Fragment Interpretation tool, which calculates the single,
Interpretation Tool non-cyclic bond cleavage fragments from a .mol file.

Table D-8 Navigation Icons on the Explore Toolbar

Icon Name Function

Open File Click to open files.

Show Next Sample Click to navigate to the next sample.

Show Previous Click to navigate to the previous sample.

Sample
GoTo Sample Click to open the Select Sample dialog.

List Data Click to view the data in tables.

Show TIC Click to generate a TIC from a spectrum.

Extract Using Click to extract ions by selecting masses.

Dialog
Show Base Peak Click to generate a BPC.
Chromatogram
Show Spectrum Click to generate a spectrum from a TIC.

Copy Graph to Click to copy the active graph to a new window.

new Window
Baseline Subtract Click to open the Baseline Subtract dialog.

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Toolbar Icons

Table D-8 Navigation Icons on the Explore Toolbar (continued)

Icon Name Function

Threshold Click to adjust the threshold.

Noise Filter Click to use the Noise Filter Options dialog to define the minimum width of a
peak. Signals below this minimum width are regarded as noise.
Show ADC Click to view ADC data.

Show File Info Click to show the experimental conditions you used to collect your data.

Add arrows Click to add arrows to the x-axis of the active graph.

Remove all arrows Click to remove arrows from the x-axis of the active graph.

Offset Graph Click to compensate for slight differences in the time during which the ADC
data and the mass spectrometer data were recorded. This is useful when
overlaying graphs for comparison.
Force Peak Labels Click to label all the peaks.

Expand Selection Click to set the expansion factor for a portion of a graph that you want to view
By in greater detail.
Clear ranges Click to return the expanded selection to normal view.

Set Selection Click to type start and stop points for a selection. This provides more accurate
selection than is possible by highlighting the region using the cursor.
Normalize to Max Click to scale a graph to maximum, so that the most intense peak is scaled is
to full scale, whether or not it is visible.
Show History Click to view a summary of data processing operations performed on a
particular file, such as smoothing, subtraction, calibration, and noise filtering.
Open Compound Click to open the compound database.
Database
Set Threshold Click to adjust the threshold.

Show Contour Plot Click to display selected data as either a spectrum graph or an XIC. Additionally,
for data acquired by a DAD, a contour plot can display selected data as either
a DAD spectrum or an XWC.
Show DAD TWC Click to generate a TWC of the DAD.

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 Toolbar Icons

Table D-8 Navigation Icons on the Explore Toolbar (continued)

Icon Name Function

Show DAD Click to generate a DAD spectrum.
Spectrum
Extract Click to extract up to three wavelength ranges from a DAD spectrum to view
Wavelength the XWC.

Table D-9 Integration Tab and Quantitation Wizard Icons

Icon Name Description

Set parameters from Uses the selected peak.
Background Region
Select Peak Uses the selected background.

Manual Integration Mode Manually integrates peaks.

Show or Hide Parameters Toggles the peak-finding parameters between shown and hidden.

Show Active Graph Shows the analyte chromatogram only.

Show Both Analyte and IS Shows the analyte and its associated chromatogram (available only
when an associated internal standard exists).
Use Default View for Graph Returns to the preset (view all data) view (if, for example, the user
has zoomed in on a chromatogram).

Table D-10 Results Table Icons

Icon Name Description

Sort Ascending by Selection Sorts the selected column by ascending values.

Sort Descending by Sorts the selected column by descending values.

selection
Lock Or Unlock Column Locks or unlocks the selected column. A locked column cannot be
moved.
Metric Plot By Selection Creates a metric plot from the selected column.

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Toolbar Icons

Table D-10 Results Table Icons (continued)

Icon Name Description

Show all Samples Shows all the samples in the Results Table.

Delete Formula Column Deletes formula columns.

Report Generator Opens the Reporter software.

Table D-11 Icon Quick Reference: Quantitate Mode

Icon Name Description

Add/Remove Samples Adds or removes samples from the Results Table.

Export as Text Saves the Results Table as a text file.

Modify Method Opens a wiff file.

Peak Review - Pane Opens peaks in a pane.

Peak Review - Window Opens peaks in a window.

Calibration - Pane Opens the calibration curve in a pane.

Calibration - Window Opens the calibration curve in a window.

Show First Peak Shows the first peak in the pane or window.

Show Last Peak Shows the last peak in the pane or window.

Show Audit Trail Shows the audit trail for the Results Table.

Clear Audit Trail Clears the audit trail for the Results Table. This functionality is not
available.
Statistics Opens the Statistics window.

Report Generator Opens the Reporter software.

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Glossary of Symbols E
Note: Not all of the symbols in the following table are applicable to every instrument.

Symbol Description
Australian Regulatory Compliance Mark. Indicates the products complies with Australian
communications Media Authority (ACMA) EMC Requirements.

Alternating current

A Amperes (current)
Authorized representative in the European community

Biohazard

CE Marking of Conformity

cCSAus mark. Indicates electrical safety certification for Canada and USA.

Catalogue number

Caution

Note: In SCIEX documentation, this symbol identifies a personal injury hazard.

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Glossary of Symbols

Symbol Description
China RoHS Caution Label. The electronic information product contains certain toxic or
hazardous substances. The center number is the Environmentally Friendly Use Period
(EFUP) date, and indicates the number of calendar years the product can be in operation.
Upon the expiration of the EFUP, the product must be immediately recycled. The circling
arrows indicate the product is recyclable. The date code on the label or product indicates
the date of manufacture.
China RoHS logo. The device does not contain toxic and hazardous substances or elements
above the maximum concentration values, and it is an environmentally-friendly product
that can be recycled and reused.
Consult instructions for use.

cTUVus mark for TUV Rheinland of North America.

Data Matrix symbol that can be scanned by a barcode reader to obtain a unique device
identifier (UDI).

Ethernet connection

Explosion Hazard

Fire Hazard

Fragile

Fuse

Hz Hertz

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 Glossary of Symbols

Symbol Description
High Voltage. Electrical Shock Hazard
If the main cover must be removed, contact a SCIEX representative to prevent electric
shock.
Hot Surface Hazard

In Vitro Diagnostic Device

Ionizing Radiation Hazard

Keep dry.
Do not expose to rain.
Relative humidity must not exceed 99%.
Keep upright.

Laser Radiation Hazard

Lifting Hazard

Manufacturer

Moving Parts Hazard

Pinch Hazard

Pressurized Gas Hazard

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Glossary of Symbols

Symbol Description
Protective Earth (ground)

Puncture Hazard

Serial number

Toxic Chemical Hazard

Transport and store the system within 66 kPa to 103 kPa.

Transport and store the system within 75 kPa to 101 kPa.

Transport and store the system within 10% to 90% relative humidity.

Transport and store the system within –30 °C to +45 °C.

Transport and store the system within –30 °C to +60 °C.

USB 2.0 connection

USB 3.0 connection

Ultraviolet Radiation Hazard

VA Volt Ampere (power)

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 Glossary of Symbols

Symbol Description
V Volts (voltage)
WEEE. Do not dispose of equipment as unsorted municipal waste.

W Watts
yyyy-mm-dd
Date of manufacture

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Glossary of Warnings F
Note: If any of the labels used to identify a component become detached, contact an FSE.

Label Translation (if applicable)

FOR RESEARCH USE ONLY. NOT FOR USE IN FOR RESEARCH USE ONLY. NOT FOR USE IN
DIAGNOSTIC PROCEDURES. DIAGNOSTIC PROCEDURES.
IMPACT INDICATOR IMPACT INDICATOR
SENSITIVE PRODUCT WARNING SENSITIVE PRODUCT WARNING

Note: If the indicator is tripped, then this container

has been dropped or otherwise mishandled. Make a
note on the Bill of Lading and then check for damage.
Any claims for shock damage require a notation.

IMPORTANT! IMPORTANT!
RECORD ANY VISIBLE CRATE DAMAGE INCLUDING RECORD ANY VISIBLE CRATE DAMAGE INCLUDING
TRIPPED “IMPACT INDICATOR” OR “TILT INDICATOR” TRIPPED “IMPACT INDICATOR” OR “TILT INDICATOR”
ON THE WAYBILL BEFORE ACCEPTING SHIPMENT AND ON THE WAYBILL BEFORE ACCEPTING SHIPMENT AND
NOTIFY YOUR LOCAL AB SCIEX CUSTOMER SUPPORT NOTIFY YOUR LOCAL AB SCIEX CUSTOMER SUPPORT
ENGINEER IMMEDIATELY. ENGINEER IMMEDIATELY.
DO NOT UNCRATE. CONTACT YOUR LOCAL CUSTOMER DO NOT UNCRATE. CONTACT YOUR LOCAL CUSTOMER
SUPPORT ENGINEER FOR UNCRATING AND SUPPORT ENGINEER FOR UNCRATING AND
INSTALLATION. INSTALLATION.
MINIMUM OF SIX PERSONS REQUIRED TO SAFELY LIFT MINIMUM OF SIX PERSONS REQUIRED TO SAFELY LIFT
THIS EQUIPMENT THIS EQUIPMENT
TIP & TELL Tilt Indicator

Note: Indicates whether the container was tipped or

mishandled. Write on the Bill of Lading and inspect
for damage. Any claims for tipping require a notation.

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 Glossary of Warnings

Label Translation (if applicable)

TiltWatch PLUS Tilt Indicator
ShockWatch
Note: Indicates whether the container was tipped or
mishandled. Write on the Bill of Lading and inspect
for damage. Any claims for tipping require a notation.

WARNING: DO NOT OPERATE WITHOUT FIRST WARNING: DO NOT OPERATE WITHOUT FIRST
ENSURING BOTTLE CAP IS SECURED. ENSURING BOTTLE CAP IS SECURED.

Note: This warning is attached to the source exhaust

waste bottle.

WARNING: NO USER SERVICEABLE PARTS INSIDE. WARNING: NO USER SERVICEABLE PARTS INSIDE.
REFER SERVICING TO QUALIFIED PERSONNEL. REFER SERVICING TO QUALIFIED PERSONNEL.

Note: Consult instructions for use.

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