Analyst® 1.

7 Software
Scheduled MRM™ Algorithm Tutorial

RUO-IDV-05-0261-E October 2017

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Contents

Scheduled MRM™ Algorithm Tutorial.......................................................................................................4

Objectives..........................................................................................................................................................................4
TM
About the Scheduled MRM Algorithm........................................................................................................................4
Related Documentation.....................................................................................................................................................5
Contact Us.........................................................................................................................................................................6
Technical Support..............................................................................................................................................................6
Prerequisites......................................................................................................................................................................7
Create a csv or txt File.......................................................................................................................................................7
TM
Create a Scheduled MRM Algorithm Acquisition Method..........................................................................................7
TM
Create an Acquisition Method using Two Scheduled MRM Algorithm Scans...........................................................15
Generate an Extracted Ion Chromatogram......................................................................................................................15
View MRM Transitions.....................................................................................................................................................17
Create Quantitation Methods..........................................................................................................................................18
Review the Results Table.................................................................................................................................................20
TM
About the Scheduled MRM Pro Algorithm...............................................................................................................22
TM
Create a Scheduled MRM Pro Algorithm Acquisition Method..................................................................................23
TM
Impact of Scheduled MRM Pro Algorithm in IDA.....................................................................................................26
TM
Create an IDA Scheduled MRM Pro Algorithm Acquisition Method.........................................................................26
TM
View the Scheduled MRM Pro Algorithm Parameters in the File Info.......................................................................27
Revision History........................................................................................................................................29

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Scheduled MRM™ Algorithm
Tutorial

Objectives
Users will learn how to:
TM
• Create a method to monitor Scheduled MRM algorithm transitions.
TM
• View Scheduled MRM algorithm transitions in an extracted ion chromatogram.
• Analyze the quantitative data by creating the quantitation method and reviewing the Results Table.
TM
• Create a Scheduled MRM Pro Algorithm Acquisition Method.
TM
• Create an IDA Scheduled MRM Pro Algorithm Acquisition Method.

TM
About the Scheduled MRM Algorithm
TM
The Scheduled MRM algorithm aids in the acquisition of hundreds of compounds based on a list of multiple
reaction monitoring (MRM) transitions, retention times, and compound IDs that you provide when creating the
TM
acquisition method. The Scheduled MRM algorithm functionality reduces the requirement for multi-period
experiments. You can also use it as a survey scan in an IDA (Information Dependent Acquisition) method.
The algorithm maximizes points across the chromatographic peak to give better peak detection and improved
reproducibility. With this feature, you can also view data files with many MRM transitions by showing the compound
ID, analyte Integration Quality™ index, and IS (internal standard) Integration Quality index columns in the Results
+ TM
Table. For the 3500, 4500, 5500, 6500, and 6500 series of instruments, a maximum of 4000 Scheduled MRM
TM
transitions are supported. For all other series of instruments, a maximum of 1000 Scheduled MRM transitions
®
are supported. Refer to the Analyst software Release Notes.

Note: The Analyte Integration Quality Index column and the IS Integration Quality Index column are also
available for MRM data in the Results Table.
TM
Figure 1 shows an example of a Scheduled MRM algorithm LC run. The number of MRM transitions monitored
simultaneously varies during the LC analysis, but remains constant between injections.

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TM
Figure 1 Typical Example of a Scheduled MRM Algorithm LC Run

Item Description
1 Monitored MRM transitions.
2 High number of MRM transitions monitored.
3 Low number of MRM transitions monitored.

TM
If you are interested in processing a large number of Scheduled MRM algorithm transitions, then use the
TM
MultiQuant software for data processing. For more information, contact a SCIEX sales representative.

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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Scheduled MRM™ Algorithm Tutorial

Contact Us
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information, visit the website at sciex.com.

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Prerequisites
Prerequisites
Users should be able to:
• Create an acquisition method
• Submit a batch
• Create a quantitation method, and create and review a Results Table.
The following peripheral devices must be included in the hardware profile:
• LC pump
• Autosampler

Create a csv or txt File

TM
Optionally, transition information for a Scheduled MRM method can be created and stored in a csv or txt file
TM
and can be imported into the mass ranges table of a Scheduled MRM Algorithm method. Use the following
criteria for creating the csv or txt file:
• The file must not contain any header, column title or row title.
• The order and number of columns in the file must match the order and number of the columns in the mass
ranges table.
TM TM
• For MRM and Scheduled MRM algorithm methods, there must be no empty cells. For Scheduled MRM
Pro Algorithm methods, the Window and Threshold columns can be left blank to allow for use of default
values. The Group column can also be left blank.
Make sure that the file is saved with either a csv or txt extension.

TM
Create a Scheduled MRM Algorithm Acquisition
Method
+
Note: For the 3500, 4500, 5500, 6500, and 6500 series of instruments, a maximum of 1250 unscheduled MRM
TM
transitions and 4000 MRM transitions with the Scheduled MRM algorithm can be acquired. For all of the
other mass spectrometers, a maximum of 300 unscheduled MRM transitions and 1000 MRM transitions with
TM
the Scheduled MRM algorithm can be acquired.

1. On the Navigation bar, under Acquire, double-click Build Acquisition Method and then, in the Acquisition
Method pane, select the Mass Spec icon.

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2. Make sure that the selected Scan Type is MRM and then select the Enabled check box in the Scheduled
+
MRM group. For the 3500, 4500, 5500, 6500, and 6500 series of instruments, the features described in
Figure 2 are used to create the method. For all of the other mass spectrometers, the features described in Figure
3 are used to create the method.

TM
Figure 2 Scheduled MRM Algorithm Software Features for the 3500, 4500, 5500, 6500,
+
and 6500 Series of Instruments

Item Description
1 Enabled check box in the Scheduled MRM group: Select to enable the basic and advanced
TM
Scheduled MRM algorithm features.
TM
2 Basic option: Select to enable the Scheduled MRM algorithm feature. Basic is the preset
option.
3 Q1/Q3 Resolution check box: Select this check box to apply different Q1 and Q3 resolution
settings to each transition. When selected, it adds the Q1 Resolution column and the Q3
Resolution column in the mass ranges table.
4 Import List button: Click to import MRM transitions, time, ID, and compound-dependent
parameters from a txt or csv file.
5 Retention Time (min) column: Type the expected retention time in minutes for the
corresponding MRM transition. This column shows the dwell time in msec for MRM methods.
6 ID column: (Optional) Type a compound ID for the transition of interest.
7 MRM detection window (sec) field: Type the amount of time for detection that surrounds
the retention time for each transition.

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Item Description
8 Target Cycle Time (across sMRM experiments) option: Select this option to specify
TM
and use the target cycle time for all of the Scheduled MRM experiments in the entire period
TM
or method. Selecting or clearing the option in one Scheduled MRM experiment automatically
TM
applies the selection to the other Scheduled MRM experiment if there are two Scheduled
TM TM
MRM experiments in the method. Maximum of two Scheduled MRM experiments are
allowed in one period or method. The target cycle time is adjustable so that a specific cycle
time can be targeted throughout all cycles to get more evenly distributed data points across a
peak regardless of a polarity switch between the concurrent transitions within a cycle. Target
Cycle Time is the preset setting.
9 Target Scan Time (per sMRM experiment) (sec) field: Select this option to specify the
target amount of time to use for the experiment in each cycle. The software will keep the total
scan time for this experiment close to the target time in each cycle unless the minimum dwell
time or the maximum dwell time is applied to some of the concurrent transitions. The target
scan time is adjustable so that a specific number of points across the LC peaks can be targeted.

TM
Figure 3 Scheduled MRM Algorithm Software Features for Other Mass Spectrometers

Item Description
TM
1 Enabled check box in the Scheduled MRM group: Select to enable the Scheduled MRM
algorithm feature.
2 Q1/Q3 Resolution check box: Select this check box to apply different Q1 and Q3 resolution
settings to each transition. When selected, it adds the Q1 Resolution column and the Q3
Resolution column in the mass ranges table.
3 Import List button: Click to import MRM transitions, time, ID, and compound-dependent
parameters from a txt or csv file.

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Item Description
4 Retention Time (min) column: Type the expected retention time in minutes for the
corresponding MRM transition. This column shows the dwell time in msec for MRM methods.
5 ID column: (Optional) Type a compound ID for the transition of interest.
6 MRM detection window (sec) field: Type the amount of time for detection that surrounds
the retention time for each transition.
7 Target Cycle Time (across sMRM experiments) option: Select this option to specify
TM
and use the target cycle time for all of the Scheduled MRM experiments in the entire period
TM
or method. Selecting or clearing the option in one Scheduled MRM experiment automatically
TM
applies the selection to the other Scheduled MRM experiment if there are two Scheduled
TM TM
MRM experiments in the method. Maximum of two Scheduled MRM experiments are
allowed in one period or method. The target cycle time is adjustable so that a specific cycle
time can be targeted throughout all cycles to get more evenly distributed data points across a
peak regardless of a polarity switch between the concurrent transitions within a cycle. Target
Cycle Time is the preset setting.
8 Target Scan Time (per sMRM experiment) (sec) field: Select this option to specify the
target amount of time to use for the experiment in each cycle. The software will keep the total
scan time for this experiment close to the target time in each cycle unless the minimum dwell
time or the maximum dwell time is applied to some of the concurrent transitions. The target
scan time is adjustable so that a specific number of points across the LC peaks can be targeted.

3. To use different Q1 and Q3 Resolution settings for each transition, click Q1/Q3 Resolution in the Scheduled
MRM group.
Two columns are added in the mass ranges table: Q1 Resolution and Q3 Resolution.

+
Figure 4 Q1/Q3 Resolution Option Selected for the 3500, 4500, 5500, 6500, and 6500
Series of Instruments

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Item Description
1 Q1 Resolution column: The mode in which the Q1 scan separates closely spaced components.
This is the resolving ability of the first quadrupole. Possible options are High, Unit, Low, or
Open.
2 Q3 Resolution column: The mode in which the Q3 scan separates closely spaced components.
This is the resolving ability of the third quadrupole. Possible options are High, Unit, Low, or
Open.

The Resolution Q1 option and the Resolution Q3 option on the Advanced MS tab are not available
for use.
If the Q1/Q3 Resolution check box is selected in Tune Method Editor, then the Q1 Resolution option
and the Q3 Resolution option on the Resolution tab are not available for use.
4. Complete the mass ranges table for each MRM transition of interest, using one of the following methods:

• Type MRM transitions manually: Type the Q1 mass, Q3 mass, retention time, and compound ID for each
transition of interest. For each transition, select values in Q1 Resolution and Q3 Resolution columns.
Right-click to add compound-dependent parameters as required. A maximum of four compound-dependent
parameters can be added to the mass ranges table.
• Import MRM transitions: Click Import List and, in the Open window, select either a csv (comma-separated
values) or txt (tab-separated values) file that contains the MRM transition information. After selecting the
file, click Open. The file contents are shown in the mass ranges table. For more information on creating
files, refer to Create a csv or txt File.
• Copy and paste MRM transitions: Select the cells containing the required information from a csv or txt file
and then press Ctrl+C. When pasting lines of information, select the first Q1 Mass (Da) cell in the mass
ranges table and then press Ctrl+V.

Note: Before importing or copying and pasting, make sure that the columns of data in the csv or txt file
match those in the mass ranges table in the software. The number of columns and column order in the source
file and destination table must be the same. Add, remove, or reorder the columns in the source file as required.
To add a column to the mass ranges table, right-click in the mass ranges table and then select a
compound-dependent parameter. For compound dependent parameters, the values must be in the allowable
ranges for the selected polarity.

5. In the MRM detection window (sec) field, type the amount of time for detection that surrounds the
retention time for each transition. This window should reflect the expected width of the chromatographic peak
and the variability in the chromatographic retention time of the analyte such that the entire MRM peak is
always in the window.
TM
Use the LC chromatography as a guide to select the best Scheduled MRM algorithm parameters. Determine
the width of a typical peak at base and then refer to Table 1 for the recommended settings. It is also important
to consider the stability of the retention time in defining the MRM detection window.

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TM
Table 1 Recommended Settings for Scheduled MRM Algorithm Parameters

Peak Width At Base MRM Detection Window Target Scan Time or Target
Cycle Time
30 seconds 90 seconds 2 seconds
15 seconds 60 seconds 1 second
10 seconds 30 seconds 0.5 seconds

TM
For the Scheduled MRM algorithm, the number of analytes per cycle monitored is adjusted based on their
retention time window. To maximize the dwell time used for each analyte and its signal-to-noise ratio, we
recommend using a smaller, but reasonable, retention time window that allows the peak of interest to be
captured. A value of 60 seconds is a good starting point. This is sufficient for chromatography that yields a
peak width of 15 seconds and a potential retention time shift of 20 seconds both to the left and to the right
of the peak.
For example, if the expected retention time is 4.5 minutes, typing 60 seconds sets a detection window from 4
minutes to 5 minutes.

Note: If the Retention Time is set to 0, the software will monitor that transition for the full run time.

6. Do one of the following as required:

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• If required, let the Target Cycle Time (across sMRM expts) option remain selected and then in the
highlighted box in the figure below, type the target cycle time. This option targets that the same cycle time
is used for each cycle, whether it is for the cycle where only one experiment or the cycle where two
experiments with polarity switches are to be run. Use the target cycle time that would consider all of the
pause times and polarity switch times if there are for each cycle. In this case, users can plan or calculate
the number of data points they could have across the run time or any detection window, and more evenly
distributed data points across a peak can be achieved.

Figure 5 Target Cycle Time (across sMRM expts)

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• If required, click Target Scan Time (per sMRM expt), and then in the highlighted box in the figure
below, type in seconds the target length of time for the experiment for each cycle. This parameter helps to
define the number of points across the chromatographic peak.

Figure 6 Target Scan Time (per sMRM expt)

Use the width of the chromatographic peaks as a guide to help set this value. A value of 1 second is a good
starting point for chromatography that yields a peak width of 15 seconds. In this case, a 1-second target
scan time will generate approximately 15 data points over a 15-second peak when there is no minimum
dwell or maximum dwell being applied to any of the concurrent transitions within this detection window.

Note: Due to the high number of concurrent transitions and minimum dwell time being applied to some
of the transitions in a cycle, an actual cycle time much higher than the target might be applied to some
cycles. Whereas, due to the low number of concurrent transitions and maximum dwell time being applied
to some of the transitions in a cycle, an actual cycle time much lower than the target might be applied
to some cycles.

7. Provide the required values in the remaining fields of the acquisition method.
8. Save the acquisition method in the project from which the acquisition will run.

TM
Note: The Scheduled MRM algorithm fields are also available in the Tune Method Editor.

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Create an Acquisition Method using Two Scheduled

TM
MRM Algorithm Scans
Use this procedure to create a method that allows for polarity switching.
TM TM
1. Create a Scheduled MRM Algorithm acquisition method using the steps in Create a Scheduled MRM
Algorithm Acquisition Method on page 7.
2. In the Acquisition method pane, right-click Period and then click Add experiment.
A second MRM scan experiment is created.
3. On the MS tab, select one of the following options based on the mass spectrometer being used:
+
• For the 3500, 4500, 5500, 6500, and 6500 series of instruments, select the Enabled check box in the
TM
Scheduled MRM group. To create a regular Scheduled MRM Algorithm acquisition method, make
sure that the Basic option is selected in the Scheduled MRM group. The features described in Figure
TM
2 on page 8 are used to create the method. To create a Scheduled MRM Pro Algorithm method, make
TM
sure the Advanced option is selected. Use Create a Scheduled MRM Pro Algorithm Acquisition Method
on page 23 to create this method.
• For all of the other mass spectrometers, select the Enabled check box in the Scheduled MRM group.
The features described in Figure 3 on page 9 are used to create the method.
TM
4. Complete the acquisition method as described in steps 3 to 9 in Create a Scheduled MRM Algorithm
Acquisition Method on page 7.
5. Save the acquisition method in the project from which the acquisition will run.

+
Note: For the 6500 series of instruments, the user-configured settling time will be applied when there is
a polarity switch in that cycle. For all other mass spectrometers, the default settling time will be applied when
there is a polarity switch in that cycle.

Generate an Extracted Ion Chromatogram

TM
Note: If a Scheduled MRM algorithm wiff file containing more than 2500 transitions is opened, a TIC is
seen instead of an XIC.

TM
1. After generating Scheduled MRM algorithm data using the acquisition method created in the previous
procedure, on the Navigation bar, under Explore, double-click Open Data File and then select the data
file and sample.
TM
MRM and Scheduled MRM algorithm data are preset to be shown as an overlaid XIC.

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2. Click Explore > Extract Ions > Use Dialog.

The Extract Ions dialog opens showing each MRM transition in the selected data file, along with the
corresponding expected retention time and compound ID, if entered.

Figure 7 Extract Ions Dialog

3. Select to sort the list by either Q1 Mass, Q3 Mass, RT (retention time), or compound ID.
4. Select one or more transitions.
5. Click OK.
The XIC is shown below the chromatogram and the Compound ID of the first selected transition is shown in
the title.

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Figure 8 Example of an Overlaid XIC that Opens When Multiple Ions are Extracted

View MRM Transitions

1. Generate an XIC.
2. Right-click the XIC title to show the MRM transitions that are active in the region (Figure 9 on page 18.) Select
the MRM transition of interest to show a label of the retention time in the chromatogram.

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Figure 9 Active MRM Transitions

3. Drag the cursor along the x-axis to zoom in on a specific time region.
The XIC now rescales to the highest peak in the shown data.
4. Right-click the XIC title again to show the MRM transitions that are active in the specific time region.
All transitions above the threshold and inside the zoomed in region are shown. The title will be reduced to the
number of transitions in the zoomed section.

Create Quantitation Methods

®
Note: In the Analyst software, you must use Build Quantitation Method to create a quantitation method for
a data file that contains more than 94 transitions. The Quantitation Wizard can only create quant methods for
data files with 94 transitions or less.

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Before you begin, make sure that you are using the recommended IntelliQuan-MQ III algorithm. For more information
on selecting algorithms, refer to the Help system.
1. On the Navigation bar, under Quantitate, double-click Build Quantitation Method.

Note: Users can use the Quantitation Wizard to create a quantitation method to analyze data that contains
less than 94 transitions.

2. Select the data file and sample you just acquired and then click OK.
A Name column is shown in the Analytes table. Because the transitions were selected during the acquisition,
this column is populated with the compound ID from the acquisition method.

Figure 10 Quantitation Method: Components Tab

Note: If you have multiple experiments, review each experiment by selecting it from the Data Source list in
order for the analytes in the experiment to be used in the quantitation method. If there are many analytes,
it might take some time for the Data Source list to be populated.

3. Set the required values in the remaining fields of the quantitation method and then save the quantitation
method.
4. Use the Quantitation Wizard to create the Results Table. Make sure to select the quantitation method that
was just created.

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Tip! If you have a large number of MRM transitions, use the Create Quan Methods From Text Files script
and the Create Text File from Quan Method script to create or modify a quantitation method. For more
information, refer to the Scripts User Guide.

Review the Results Table

1. To view the results for a specific transition, right-click in the Results Table, select Analyte and then select
the transition from the list of compound IDs.

Figure 11 Results Table: Analyte Selection

2. Right-click in the Results Table and then click Table Settings > Edit to open the Table Settings dialog.
3. Double-click Columns and then select Analyte from the list.
4. Beside Analyte Peak Name, select the Shown check box.
5. Click OK and then click Done.
The Analyte Peak Name column is added to the Results Table and the compound ID of each transition is
shown in that column.

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Figure 12 Results Table: Analyte Peak Name Column

6. Right-click in the Results Table and then click Table Settings > Edit to show the Analyte Integration Quality
and IS Integration Quality columns in the table.
7. Double-click Columns and then select Analyte from the list.
8. Beside Analyte Integration Quality, select the Shown check box, and then click OK.
9. Select Internal Standard from the list.
10. Beside IS Integration Quality, select the Shown check box.
11. Click OK and then click Done.
The two columns are added to the Results Table. Integration quality indicates how well the peak is integrated.
Values closer to 1 indicate well-integrated peaks. Smaller values can indicate that the peak is not well-integrated,
that there is a large background, or that there might be another peak in the region.
These columns facilitate peak review as you can easily see the peaks with low analyte Integration Quality™
index values for manual review. In addition, you can query the data for the analyte Integration Quality index
values that are less than a value you consider acceptable to show and manually review a subset of the data.

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Figure 13 Results Table Columns

Item Description
1 Analyte Integration Quality Index column
2 IS Integration Quality Index column

TM
About the Scheduled MRM Pro Algorithm
TM +
The Scheduled MRM Pro Algorithm feature is supported in the 3500, 4500, 5500, 6500, and 6500 series of
instruments.
TM TM
The Scheduled MRM Pro Algorithm adds advanced functionality to the Scheduled MRM Algorithm. It
improves the retention time robustness of experiments by allowing acquisition windows to be set for each transition
in the acquisition method. Users can adjust individual windows for compounds that have broad LC peaks or large
variation in their retention times.
TM
The Scheduled MRM Pro Algorithm also supports automatic window extension during acquisition. Users have
the option of turning Dynamic Window Extension (DWE) on or off. Users can also set different trigger thresholds
and DWE thresholds for each individual transition, such as a low trigger threshold and a high DWE threshold for
each transition which triggers more secondary transitions but avoids unnecessary dynamic window extensions.
When DWE is enabled, if a compound has shifted to a later retention time and the intensity has not dropped below
TM
its extension threshold past its retention time, then the Scheduled MRM Pro Algorithm will automatically
extend the detection window until its intensity drops below the threshold. The transition will be monitored for
another half of the method specified detection window. The detection window after the extension is up to double
the specified acquisition window. This feature allows smaller windows to be used, but at the same time makes
sure that every peak is captured in its entirety. This feature increases method robustness to most shifts in retention
time.
TM
The Scheduled MRM Pro Algorithm supports the labeling of multiple transitions for an analyte as either primary
or secondary. Primary transitions are monitored throughout the acquisition window, while secondary transitions

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are only monitored after the associated primary transitions reach their trigger thresholds. This minimizes the cycle
time by reducing the number of MRM transitions monitored. The acquisition time is focused on collecting data for
the analytes that are present in a sample and not for analytes that are not present in the sample. The Scheduled
TM
MRM Pro Algorithm also supports the use of dwell weight. Dwell weight allows required dwell time to be
expressed relatively. Highly abundant compounds can be assigned a low dwell weight, while less abundant
compounds can be assigned a high dwell weight. During the run, the available dwell time will be assigned based
on this weighting.
TM
The Scheduled MRM Pro Algorithm also supports Dynamic Background Subtraction (DBS) for triggering the
TM TM
secondary Scheduled MRM transitions. The DBS option for Scheduled MRM experiments is only available
TM TM
to non-IDA Scheduled MRM Algorithm methods. For non-IDA Scheduled MRM methods or experiments,
TM
when DBS is enabled, it is applied to the primary Scheduled MRM transitions for triggering secondary
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Scheduled MRM transitions in that experiment. For IDA methods with Scheduled MRM algorithm as survey
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scans, if DBS is enabled in the IDA Criteria, then it is applied to the primary Scheduled MRM transitions for
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both triggering the secondary Scheduled MRM transitions and triggering dependent scans. When both DWE
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and DBS are enabled for the Scheduled MRM experiment, DWE triggering depends on the non-processed data
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without DBS of the primary transitions. With DBS enabled for a Scheduled MRM experiment, the secondary
transitions, once triggered, will continue to acquire till their primary transitions stop acquiring regardless DWE
has occurred.

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Create a Scheduled MRM Pro Algorithm
Acquisition Method
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The Scheduled MRM Pro Algorithm feature is only supported in the 3500, 4500, 5500, 6500, and 6500+ series
of instruments.
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Use this procedure to create a Scheduled MRM Pro Algorithm method in the Method Editor window. This type
of method can also be created in the Manual Tune window.
1. Activate a hardware profile for the supported mass spectrometer. For 6500 and 6500+ series of instruments,
if required, select a different mass mode in the hardware profile and then activate the hardware profile.
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2. Open an existing Scheduled MRM Algorithm acquisition method or create one using the procedure Create
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a Scheduled MRM Algorithm Acquisition Method on page 7.
3. Select the Advanced option in the Scheduled MRM group on the MS tab in the Acquisition Method Editor.
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This enables the Scheduled MRM Pro algorithm feature.
Five new columns are added in the mass ranges table and two check boxes, Dynamic Window Extension
and Dynamic Background Subtraction are shown in the Scheduled MRM group.

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Figure 14 Scheduled MRM Pro Algorithm Parameters

Item Description
1 Group: Use this column to group the transitions of a compound together. Assign the same
name to all the transitions that are part of one group.
2 MRM Window (sec): Specify the length of time, centered around the retention time, that
the transition will be monitored. Values for transitions in this field supersede the method
default window in the MRM detection window field. The Window column is similar to the
MRM detection window field but the value entered only applies to that specific transition. If
left blank, the value is taken from the MRM detection window field.
3 Primary/Secondary: Use this column to specify whether a transition is primary or secondary.
Primary transitions are monitored for the entire acquisition window. Secondary transitions are
monitored within the acquisition window as long as all the primary transitions are above their
individual thresholds.
Assign the number '1' to the primary transitions and '2' to the secondary transitions in a group.
There can be multiple primary and secondary transitions in a group.
4 Trigger Threshold: Specify the intensity that triggers secondary transitions. In IDA methods,
Trigger Threshold is also used to trigger dependent scans.
This value can be zero or higher.

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Item Description
5 Dwell Weight: Use this column to specify if a specific transition should have a longer scan
time (dwell time) than other transitions. The default value is 1.
Dwell weight allows the required dwell time to be expressed relatively. Highly abundant
compounds can be assigned a low dwell weight (<1), while less abundant compounds can be
assigned a high dwell weight (>1). The allowed dwell weight range is 0.1 – 10. During the
run, the available dwell time is assigned based on this weighting.

Note: The total dwell weight equals to the sum of dwell weights of all of the primary
transitions and half of the sum of the dwell weights of all of the secondary transitions in that
cycle. However, the dwell time allocation still uses their individual dwell weight divided by
the total dwell weight. For example, the dwell weight is 1 for both primary and secondary
transitions for a method containing only two transitions with the same retention time. The
total dwell weight is 1 + 0.5 = 1.5. The dwell time for both transitions will be the total dwell
time that can be assigned to all of the concurrent transitions multiplied by 1/1.5. The total
dwell time that can be assigned is calculated after deducting the pause times, polarity switch
times if applicable, and then corrected by a factor of 1.2. All blank entries for dwell weight
use the default 1.

Note: When the calculated dwell time is not the same for all of the cycles for a transition
throughout its retention time window, then the average dwell time of all cycles will be used
for acquisition.

6 Dynamic Window Extension: Select this check box to enable dynamic window extension
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in the Scheduled MRM Pro acquisition method. To not use dynamic window extension,
uncheck Dynamic Window Extension (DWE) check box. When DWE is selected, a new column
Extension Threshold is added to the mass ranges table. If DWE is selected and if the peak
intensity of a transition is still above the Extension Threshold past its retention time, the
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Scheduled MRM Pro Algorithm automatically extends the detection window until the
intensity drops below the Extension Threshold and the transition will be monitored for another
half of the method specified detection window. The acquisition window after the extension
can be up to double the method specified detection window.
7 Dynamic Background Subtraction: Select this check box to use dynamic background
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subtraction in the Scheduled MRM Pro acquisition method. In Scheduled MRM Pro
triggered IDA method, the Dynamic Background Subtraction (DBS) check box is not available
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in the Scheduled MRM Pro experiment if it is enabled in the IDA criteria. The DBS check
box in the IDA criteria, if checked, is used to trigger both secondary transitions and dependent
scans.
8 Extension Threshold: Specify the threshold value for the dynamic window extension in
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the Scheduled MRM Pro acquisition method. This value can be zero or higher. Refer to
Figure 15.

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Figure 15 Dynamic Window Extension Selected - Extension Threshold Column

4. Complete the mass ranges table for each MRM transition by providing appropriate values in the five Scheduled
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MRM Pro Algorithm columns. Use the information in Figure 14 and the following rules:

• All the transitions belonging to the same group must be listed consecutively in the mass ranges table.
• All the transitions in the same group must have the same retention time in the Retention Time (min) column.
• All the transitions in the same group must have the same time in the MRM Window (sec) column.
• All the primary transitions must be entered before the secondary transitions in a group. If a group has only
one transition, then that transition must be the primary transition.

5. Save the acquisition method.

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The Scheduled MRM Pro Algorithm acquisition method can only contain one period and up to two
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Scheduled MRM experiments.

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Impact of Scheduled MRM Pro Algorithm in IDA
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When Information Dependent Acquisition (IDA) survey scan is performed using the Scheduled MRM Pro
Algorithm, a dependent scan in the IDA method is only triggered when the intensities of all of the MRM transitions
in a group are above their trigger thresholds. This improves the cycle time by eliminating false triggers of dependent
scans.

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Create an IDA Scheduled MRM Pro Algorithm
Acquisition Method
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1. Create a Scheduled MRM Pro Algorithm using the procedure Create a Scheduled MRM Pro Algorithm
Acquisition Method on page 23.

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2. If required, to add an experiment of ER scan type, add it before adding the IDA Criteria.
3. Right-click the Period icon and then click Add IDA Criteria Level.
4. Specify the IDA Criteria parameters. Refer to the Information Dependent Acquisition Tutorial.
5. Right-click the Period icon and then click Add experiment.
6. On the MS tab, in the Scan type list, select a dependent scan type. For this example, select Product Ion
(MS2) or Enhanced Product Ion (EPI).

Note: For all dependent scan types, the Product Of must be 30 Da.

7. If required, copy or add more dependent experiments.

It depends on the IDA criteria, from X to Y most intense ions.

8. Specify the experiment parameters.
9. Save the acquisition method in the project from where the acquisition will run.

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Note: During data acquisition using an IDA Scheduled MRM Pro Algorithm method, the trigger threshold
for each MRM transition in the method is used to trigger dependent scans instead of the IDA threshold.

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Note: To optimize a Scheduled MRM method for the dwell time allocation, concurrency, and projected
cycle time, install and use the sMRM Calculator script. For more information, refer to the Scripts User
Guide.

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View the Scheduled MRM Pro Algorithm
Parameters in the File Info
After the acquisition of a sample is complete, the file information for the data file acquired with the Scheduled
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MRM Pro Algorithm method shows all of the user-defined parameters for each transition. These parameters
include the following: MRM being scheduled or not, Q1, Q3, Retention Time, ID, Group, MRM Window (sec),
Primary/Secondary, Trigger Threshold, Extension Threshold (if the Dynamic Window Extension check box was
selected), Dwell Weight, Q1 resolution, and Q3 Resolution (if the Q1/Q3 Resolution check box was selected). The
Dynamic Background Subtraction, Dynamic Window Extension, Dwell Time, Target Scan Time or Target Cycle Time
parameters are also shown.
1. On the Navigation bar, under Explore, double-click Open Data File.
The Select Sample dialog opens.
2. In the Data Files pane, select the wiff file.

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3. In the Samples pane, select the sample.

4. Click OK.
The data acquired from the sample opens.
5. To view the file information, click the Show File Info icon on the toolbar.
The File information pane opens below the graph.
6. Expand the required period in the left pane of the File Information pane.
7. Click the required period experiment link.
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The values for the Scheduled MRM Pro Algorithm parameters for each transition are recorded under the
selected Period Experiment section in the right pane.
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Figure 16 Scheduled MRM Pro Algorithm Parameters in File Info

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Revision History

Revision Reason for Change Date

A First release of document. April 2013
B • Updated the criteria for creating .CSV or.txt file for May 2013
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Scheduled MRM Pro algorithm method.
• Updated the description of Target Scan Time field.
• Changed the case to lowercase for MRM detection window
field.
• Modified the description of the Threshold field.
• Removed a sentence from the Impact of Scheduled
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MRM Pro Algorithm in IDA section

C Added reference to 3500 series of instruments where required June 2014

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Revision History

Revision Reason for Change Date

D • Changed AB SCIEX to SCIEX where required. August 2015
• Updated the copyright page.
• Added reference to 6500+ series of instruments where
required.
• Added Revision History.
• Added “and the minimum dwell time for each transition”
in the second note on page 9.
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E • Added the new Scheduled MRM Algorithm and October 2017
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Scheduled MRM Pro Algorithm features.
• Added new screen shots.
• Added the Contact Us topic. Removed "for more than 94
Transitions" from the Create Quantitation Methods title.
• Added a note about using the Build Quantitation Method
to create a quantitation method for a data file that contains
more than 94 transitions.
• Added a tip about using scripts in case of large number of
MRM transitions.
• Updated the content in the Objectives, About the
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Scheduled MRM Algorithm, and Related
Documentation sections.
• Updated the content in the Create an Acquisition Method
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using two Scheduled MRM Algorithm Scan and Impact
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of Scheduled MRM Pro Algorithm in IDA sections.
• New templates were applied to the content, which has led
to some edit changes in the content.

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