Analyst® 1.6.

1 Software

Automatic Optimization Tutorial

Release Date: March 2012

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Automatic Optimization Tutorial

Topics in this tutorial:

• Objectives on page 3
• About Automatic Optimization on page 3
• Related Documentation on page 4
• Technical Support on page 4
• Prerequisites on page 4
• Automatically Optimizing for an Analyte Using Infusion on page 5
• Automatically Optimizing for an Analyte using FIA on page 11

Objectives
You will learn how to:
• Automatically optimize for the analyte using the Compound Optimization wizard.
• Choose between infusion and FIA (flow injection analysis),
• Use an infusion method to optimize compound-dependent parameters.
• Use an FIA method to optimize compound- and ion source-dependent parameters.

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 displayed 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, you 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.

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Use infusion optimization to optimize compound-dependent parameters only at much lower flow
rates than those used during LC/MS analysis.

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, that is, one
compound- dependent parameter is optimized first followed by the next compound-dependent
parameter. It optimizes for ion source-dependant parameters by making one injection for each
value.
Compound parameters need to 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 1-1 Differences Between Sample Introduction Methods
Method Required devices Parameters Typical flow rate range
Infusion Syringe pump Compound-dependent 5 to 25 µL/min
FIA LC pump and Source- and compound- 25 to 1000 µL/min
Autosampler dependent

During optimization, a text file is generated and then displayed during the optimization process.
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.

Related Documentation
• Getting Started Guide for the Analyst® software
• Advanced User Guide for the Analyst software
• The Analyst software Help system
• The applicable ion source guide

Technical Support
AB SCIEX and its representatives maintain a staff of fully-trained service and technical
specialists located throughout the world. They can answer questions about the instrument or any
technical issues that may arise. For more information, visit the Web site at www.absciex.com.

Prerequisites
• The instrument is tuned and calibrated.
• An acquisition method.

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• If the system has an integrated syringe pump, there is a hardware profile containing
a syringe pump.
• For sample introduction by FIA, the hardware profile must contain an LC pump and
an autosampler.
• All the required peripheral devices, including a syringe pump, if needed, and LC
components.
The following equipment and solutions are suggested. You can use other solutions depending on
your system, peripheral devices, and experiment.
• A syringe, preferably a 1.0 ml. syringe.
• Mobile phase: 1:1 acetonitrile:water + 2 mM ammonium acetate + 0.1% formic acid.
• LC pump and autosampler.
• Autosampler vials.
• 4-compound mix, consisting of reserpine, minoxidil, tolbutamide, and rescinnamine.
The solution can be used for infusion and FIA. The concentration is system-
dependent. Use a solution that is 49.9% acetonitrile, with 50% deionized water and
0.1% formic acid as diluent.
Table 1-2 Compounds and Molecular Weights
Compound m/z
Minoxidil 210.2
Tolbutamide 271.3
Reserpine 609.4
Rescinnamine 635.4

Table 1-3 Estimates For Starting Concentrations

System Concentration
API 2000™ and QTRAP® systems 500 ng/mL
API 3200™ and 3200 QTRAP systems 50 ng/mL
API 4000™ and 4000 QTRAP systems 10 ng/mL
API 5000™ system 10 ng/mL
AB SCIEX QTRAP 4500 system 10 ng/mL
AB SCIEX Triple Quad™ 4500 system 10 ng/mL
AB SCIEX QTRAP 5500 system 10 ng/mL
AB SCIEX Triple Quad 5500 system 10 ng/mL

Automatically Optimizing for an Analyte Using

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

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Topics in this section:

• Confirming the Presence of Compounds on page 6
• Compound ions on page 8
• Checking the Optimization Results on page 11

Confirming the Presence of Compounds

You should confirm the presence of compounds of interest before continuing with automatic
optimization.
1. Create a project.
2. Activate the hardware profile.
3. Infuse the compound in solution at a rate of 5 to 10 µL/min.
4. On the Navigation bar, under Tune and Calibrate, double-click Manual Tuning.
5. If your instrument has an integrated syringe pump, in the method list field, click
Syringe Pump Method.
6. On the Syringe Pump Method Properties tab, type the parameters from the
following table.
Table 1-4 Syringe Pump Method Properties Tab
Parameter Value
Syringe Diameter Syringe dependent. 1.0 ml syringe is 4.610 mm
Flow Rate 10
Unit µL/min

Figure 1-1 Syringe Pump Method Properties tab

7. Click Start Syringe Pump.
8. In the method list field, click MS Method.
9. On the MS tab, type the parameters from the following table.

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Table 1-5 MS Tab

Parameter Value
Scan type Q1 MS (Q1)
Start (Da) 200
Stop (Da) 700
*Time (sec) (if available) 2.5
*Scan rate (Da/s) (if available) 200
Duration (min) 3
* These fields are instrument-dependent.

10. Click Start.

11. Wait until you see an even TIC on the left and peaks on the right and then click Stop.
12. Select the MCA check box and then, in the Cycles field, type 10.
13. Click Start.
14. When the ten scans are complete, you should see 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 you can not see any noise peaks. In
the first case, you may not be looking at a real compound. In the second
case, you may have too high a concentration for the software to optimize
properly.

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Figure 1-2 Compound ions

Performing 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 optimizes the
compound-dependent parameters to get the maximum sensitivity for the compound. The
software ramps CE and picks the most intense fragments meeting all the product ion selection
criteria.
If the Initial Q1 scan signal is too high, the Analyst® software attempts to lower the CEM to keep
ions within detector range. If the signal is still too high after lowering the CEM, the process stops
and an error message is displayed. You must 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. On the Navigation bar, under Tune and Calibrate, double-click Compound
Optimization.
2. On the Instrument Settings page, in the Inlet section, click Infusion.
3. In the Mass Spectrometer section, click MS/MS Analysis and then click Next.
4. On the Ions to use in MS/MS Analysis page, type the parameters from the
following table.

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Table 1-6 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
search window that you specified. If the most intense peak in that window is
not the mass of interest, the software will optimize on the wrong ion.

5. Click Criteria next to the Auto Select option.

6. In the Product Ion Auto Selection Criteria dialog, type the parameters from the
following table.
Table 1-7 Product Ion Auto Selection Criteria Dialog Parameters
Parameter Value Description
From the Most 3 The number of fragment peaks to be optimized.
Intense (peaks) The algorithm will generate a product ion scan
spectrum while ramping the CE in MCA mode. In
this example, it will then take the 3 most intense
fragment ions from the spectrum and continue
the MS/MS optimization on those fragments only.
Build final method 2 The number of fragment ions per precursor ion
using (most intense (target compound) to be automatically included in
peaks) the acquisition method. The specified number
defines the number of MRM transitions to be
included for each target compound in the
method, and the order of preference is based on
the intensity of the fragment ion.
Two is a better starting value than one because
you will typically want two product ions for
quantitation. You can start with three in case
there is a problem with one of the two best; you
can go back and the third is already identified.

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Table 1-7 Product Ion Auto Selection Criteria Dialog Parameters (Continued)
Parameter Value Description
Exclude Product 20.000 The Da value that defines the exclusion window
Ions within ± (Da of around the precursor ion so that fragment ions
Precursor Ion m/z) that fall within this window are not selected for
MRM optimization. For example, if you type ± 5
Da for a precursor ion of 500 m/z, any fragment
ions within the 495 to 505 m/z region are
excluded. This prevents the precursor ion from
being optimized.
Min. Mass for 60.000 The lowest fragment mass to be considered for
Product Ion (amu) optimization. Use 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
Product Ion (cps) be considered.

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

8. Click Next.
9. In the Target Components dialog, type the parameters from the following table.

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

transition.

Table 1-8 Target Compounds Dialog Parameters

Target Compound Field Value*
Reserpine Compound Name Reserpine
MW (Da) 609.4
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
* You must type the exact ion mass.

10. Click Finish to begin the optimization process.

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The screen displays two windows, a text file window and an acquisition window. You
may need to minimize one of them to see the other. The experiment being run is
displayed 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.

Checking 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 Method
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 select the Reserpine_QOpt_FinalMRM.POS.dam file you just
created.
3. Compare the values in the text file to those in the .dam file.
4. Check the contents of the following folders:
• Data: Look through all 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) displayed during the optimization process.

Automatically Optimizing for an Analyte using

FIA
Before you optimize using FIA, you should already have identified the ions for the compounds
and saved the basic acquisition method using the infusion method used earlier in this tutorial.
Because using FIA for optimization requires that an autosampler and LC pump be active in the
hardware profile, you must add these two devices to the basic acquisition method.

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

Before you begin, make sure that you create an LC/MS/MS acquisition method based on the
Reserpine_QOpt_FinalMRM.POS.dam file and name the new method
FIA_AutoOpt_Tutorial.dam. Make sure that you are in the project that contains the acquisition
method that you are going to work with.
1. Put a dilution of 4-compound mix in an autosampler.

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You need enough sample to look at each variable of each parameter and have
sample left over. For example, for temperature, if you want to run at 300, 400, and
500°C you need more then 30 µL (3 × 10 µL injection).
2. Confirm that LC Sync is selected in the method.

Figure 1-3 Acquisition method with LC Sync selected

3. Make sure you have reasonable starting ion source and gas parameters so you do
not contaminate the instrument while optimizing. For more information, see the
appropriate ion source operator’s guide.
4. Set the horizontal micrometer to 5 mm. Set the vertical micrometer on the ion source
for your flow rate. For the Turbo V™ ion source use the following parameters as a
starting point. For more information, see the Turbo V™ Ion Source Operator’s Guide.
Table 1-9 Turbo V Ion Source Vertical Parameters
Flow rate Initial vertical parameters
1 to 20 µL/min 10 mm
20 to 250 µL/min 5 mm
250 to 500 µL/min 2 mm
500 + µL/min 0 mm

5. Set the values for your LC 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 set 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 your compounds of interest elute.
6. On the Navigation bar, under Tune and Calibrate, double-click Compound
Optimization.
7. On the Instrument Settings page, depending on the stack you are using, type the
parameters from the following table.

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Table 1-10 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

8. Click Next.
9. On the FIA Target Compounds page, do not select the Int. Std. check box.
Selecting the check box indicates which MRM corresponds to the internal standards.
Internal standards are not optimized during the optimization process.
10. In the Resolution section, select Unit in both the Q1 Resolution and Q3 Resolution
fields.

Figure 1-4 Q1 and Q3 Resolution fields

11. Click Next.
12. 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 your system clean. The following parameters
should work for most flow rates.

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Table 1-11 Parameters for FIA Source Parameters Page

Parameter Select the Optimize Values for
check box? optimization
Curtain Gas Yes 20;40;55
Collision Gas No —
IonSpray Voltage Yes 1500;2000;3000;4000;500
0
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 —

13. 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 may
be high depending on how many variables for each parameter you are optimizing, as
each variable is a separate method.

Figure 1-5 Replicate Injection for each Parameter field

14. Click Next.
15. On the FIA Compound Parameters page, type the parameters from the following
table.

Note: The values in Table 1-12 are suggested values. For more
information, see the documentation or Help.

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Table 1-12 FIA Compound Parameters Page

Parameter Select the Optimize Values for
check box? optimization
Declustering Potential Yes 60;80;100;120;200
Entrance Potential No —
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 that will prevent proper evaluation of
the parameter.

16. In the Mass Spec. Duration field, type 1.5. This value should be at least the
required length of time for each injection.

Figure 1-6 Mass Spec. Duration field

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17. 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.
To obtain optimized parameters, this routine must be repeated. Typically, the ion
source and gas parameters must be narrowed using one more FIA cycle.
18. The software generates several acquisition methods. Open the final optimized FIA
method called *_FIA_sample_1. Save this method using a simpler name.

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