Varian, Inc.

2700 Mitchell Drive

Walnut Creek, CA 94598-1675/USA

MS Workstation

MS Data Handling
Users Guide

©Varian, Inc. 2004 Printed in U.S.A. 03-954038-00:Rev. 1

Contents

About Data Handling ................................................................................................................3

Acquire Data ...............................................................................................................................................................3
Build a Data Handling Method....................................................................................................................................3
Build Spectrum List.....................................................................................................................................................6
Peak Identification ......................................................................................................................................................7
Import Compound List ..............................................................................................................................................10
Identify Internal Standards........................................................................................................................................11
Examine Spectra ......................................................................................................................................................12
Adjusting Integration Parameters .............................................................................................................................13
Setting Up Search Parameters for Target Peak Identification .................................................................................14
Edit Reference Spectrum .........................................................................................................................................14
Fine Tuning of Integration Parameters .....................................................................................................................16
Generating Calibration Curves .................................................................................................................................17
Building A RecalcList ................................................................................................................................................17
Process / Review RecalcList ....................................................................................................................................19
Modifying a Method ..................................................................................................................................................22

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About Data Handling
Data Handling encompasses the procedures and tasks performed after the Mass
Spectrum has been collected. Depending on the Methods you develop, you may
or may not know the best type of Data Handling that should be built into the
method. Often, after running a sample and observing the chromatogram and
spectrum, it is easier to develop data handling methods.
There are four major processes that lead up to and define the development of
Data Handling methods: 1. Acquisition of data, 2. Display of data, 3. Building a
Compound List, and 4. Building a Data Handling Method.

Acquire Data
Once the chromatographic and mass spectrometric methods have been
developed, prepare a series of standard solutions of the compounds of interest,
at up to ten levels covering the range of concentrations for which the method will
be used. If you are using one or more internal standards, be sure that the
concentration of the internal standard(s) is the same as that in the midpoint of the
expected sample concentration range.
At this point you should make at least two injections of your standards: a mid-
level standard which will be used to identify the component peaks, and a low-
level standard which will be used to optimize the peak detection and identification
parameters. Of course, you may inject all levels of the standards at this time and
recalculate the calibration data after the data handling method has been
developed.

Build a Data Handling Method

The display of data, building of a Compound List, and building a Data Handling
Method will be the focus of the remaining portion of this Users Guide.

Begin by opening the Method Builder application by clicking on the icon

in the MSWS toolbar. Click on “Open an Existing Method File” and “OK”.

Find the method file to which you wish to add the Data Handling method (usually
this will be the acquisition method) and open it.

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The Method Builder application window will appear with your chosen method
(see next screenshot).

In the left area, select Calculations Setup in the MS Data Handling section.
For the purposes of this exercise, we will select Area for Measurement Type,
Internal Std for Calibration Type, and Absolute for Ion Ratio Type. Make the
appropriate changes in the following dialog:

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 About Data Handling
Build a Data Handling Method

In the left area of the window, select Compound Table in the MS Data Handling
section. You will now see an empty Compound Table in the right section. Click
on the Select Data File button and select the middle level standard file you
generated earlier. The name of the selected file will appear in the dialog. If you
know which peaks in the selected file correspond to your standard compounds,
you may now click on the Build Compound List button to begin the identification
of the compounds in your standards.

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Build Spectrum List
Clicking on the apex of the peaks will display the corresponding spectrum and
add the spectrum to a SpectrumList having the same name as the data file, e.g.,
ei 200.msp. You may also build the SpectrumList automatically by selecting
“Build SpectrumList from Active Chromatogram Plot” from the SpectrumList
menu in MS Data Review. Adjust the Peak Width, Slope Sensitivity, and Peak
Size Reject parameters so that all of your compounds of interest will be
integrated while most of the extraneous peaks will be rejected. Click on OK to
integrate the chromatogram and generate the SpectrumList.

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 About Data Handling
Peak Identification

You should now have a spectrum list with the same name as your data file, with
an entry for each peak integrated using the parameters you entered.

Peak Identification
The next step is to identify these peaks, particularly those which correspond to
your standard compounds. This is accomplished by library searching the
SpectrumList. Click on the “Library Search SpectrumList” icon in the
SpectrumList toolbar, or select “Library Search a SpectrumList” from the
SpectrumList menu. Either action will display a dialog which allows you to edit
the library search parameters.

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When you have edited these parameters, click on the Search button to begin the
identification of your peaks. The spectrum list will be updated with the name of
the best match for each peak found in the library, along with the match quality
values. Some of the peaks may be due to matrix or column bleed, and these

entries may be deleted by clicking on the entry and then on the icon. In
other cases, the best match found may not represent the actual compound in the
standard. For example, the pesticide Demeton-S gives the best match, but we
know that the isomer Demeton-O was actually included in the standard.

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 About Data Handling
Peak Identification

Selecting this line and then clicking on the “Library Search Spectrum” icon will
display the complete list of matches for the selected peak. Click on the second
line in the list, containing the correct identification, then click on the Replace

Match in Spectrum List Window icon, , to update the SpectrumList with

the corrected identification. Continue to edit the SpectrumList until you have
correctly identified all the compounds in your standard, then click on the Update
All Searches with Matches icon to complete the construction of the
SpectrumList.

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Import Compound List
Close MS Data Review and return to the Method Builder application. Click on
Import Compound List and select the .msp file you just completed.

All the identified peaks will be entered in the Compound Table along with default
values for the data handling parameters.

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 About Data Handling
Examine Spectra

Identify Internal Standards

The first step in completing the data handling method is to identify the internal
standard(s), assuming you are using an internal standard method. Double click
on the line in the compound table containing the IS information, (in our example
we will use the benzophenone peak as our IS), and select the Internal Standard
button in the Compound Type section of the Compound Attributes tab.

If you like, you can designate the internal standard peak as a Reference peak
and/or a Relative Retention peak to assist in identifying analyte peaks. It is a
good idea to use only Internal Standard peaks as Reference or Relative
Retention Time peaks, as both IS and Reference peaks are required to be
present and easily identifiable in every chromatogram for proper data handling to
occur.

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Examine Spectra
Next click on the Quan Ions tab and examine the sample and reference spectra.

The default Quan ion is the most intense ion in the sample spectrum, but you
may wish to select another ion if you know that there may be matrix interference
with the default ion. If you wish to use qualifier ions, you can add them by
clicking on the Load button in the Qualifiers section. This will load the most
intense non-quan ions into the table and calculate their abundance relative to the
selected quan ion. The method of calculating relative abundance limits for the
qualifier ions (Relative or Absolute) is selected in the Calculations Setup section
of the method; the percentage limits are entered on an ion by ion basis in this
table. You may add or delete ions as you like to arrive at a suite of ions which
are indicative of the presence of the compound of interest. For instance, you
may want to select a less intense, higher m/z ion in preference to a more intense
lighter ion which might be more likely to be interfered with by matrix components.
Select the Calculations tab and enter the # Calibration Levels you will use, in
our case six levels, and the Curve Fit Type, the Origin treatment, and the
Regression weighting formula.

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 About Data Handling
Adjusting Integration Parameters

Adjusting Integration Parameters

Since we are still working with the IS, we enter a value of 1.000 for the Cal Level
Amounts for each level. Now select the Integration tab and adjust the integration
parameters if necessary.

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Since the Internal Standard is generally present in easily detected quantities in
every chromatogram, it is probable that the default values will work quite well. If
desired, you can invoke filtering of the peak data by clicking on the Filter Peak…
button and setting the filter parameters as you like. You can test the effects of
any changes you make in the integration tab by clicking on the Integrate button
and observing the effect on the Peak Area and Integrated Peak display.

Setting Up Search Parameters for Target Peak

Identification
The Identification tab is used to set up the search parameters for target peak
identification.

You can specify the Search Window time range within which the target must be
found, the Search Type (Spectrum or Retention time), the minimum required
Match Threshold, and the Minimum Abundance to be considered in
calculating a match.

Edit Reference Spectrum

In the Ref. Spectrum tab, you can edit the reference spectrum to improve the
match reliability, and, if necessary, use a sample spectrum as the reference, for
those cases where the compound of interest is not found in the library, or where
the MS conditions do not allow matching to a standard library.

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 About Data Handling
Edit Reference Spectrum

For Target compounds, the editing of the Compound Table is similar to that for
the Internal Standard, except of course the compound is identified as an Analyte
in the Compound Attributes tab, and the appropriate Internal Standard is
selected. In the Calculations tab, the Calibration Curve Calculations are changed
to match the IS, and the Calibration Level Amounts are changed to reflect the
levels found in the calibration standards.

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Fine Tuning of Integration Parameters
Finally, to fine tune the Integration parameters, close the target compound dialog,
click on Select Data File, and select the file for the lowest level calibration
standard. Double click on the first target compound in the Compound List and
select the Integration tab.

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 About Data Handling
Building A RecalcList

Examine the integrated peak display and adjust the integration method until the
peak is processed as you would like when you press the Integrate button. You
may need to use Timed Events to encourage the software to perform the
appropriate integration. Repeat the process for each of the remaining target
compounds. Once you have found integration parameters which work for most
or all of the compounds, you may wish to use these same parameters to
integrate unknown peaks. If so, select the Calculations Setup section of the
method, check the Report Unknown Peaks box, and modify the parameters in
the Chromatogram Processing section as required.

Generating Calibration Curves

Once all the target compounds have been properly identified and the integration
parameters have been optimized, calibration curves for all the compounds of
interest may be generated. If the data files for all the replicates of all the
calibration levels were generated by execution of a SampleList, a RecalcList
probably already exists which can be used to generate the calibration curves.

Building A RecalcList
You will need to build a RecalcList if:
• The injections were made individually.
• The injections were made from a number of different SampleLists.
• You chose not to automatically generate a RecalcList.

Click on the “Edit Automation Files” icon in the MS Workstation Toolbar

and select either File:New or File:Open and then RecalcList… to access either a
new RecalcList or an existing RecalcList. If you are generating a new RecalcList,
select the folder in which the calibration files currently reside and give the
RecalcList a meaningful File name.

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When the new RecalcList appears, begin by adding a line and selecting New
Calibration Block as the Sample Type. This will ensure that any recalculation
is independent of previous actions. Next add another line and click on the Data
File field, then click on the Browse… button.

A list of data files will appear. Select the first replicate of the lowest level
standard and click on Open to add the filename to the RecalcList. Change the
Sample Type to Calibration and the Cal Level to whatever it is labeled in the
Compound Table (it doesn’t matter whether you use 1 as the lowest level
standard or the highest level standard, as long as the RecalcList and the
compound table agree). Add any notes and edit any of the other fields in the line
as necessary. Add any replicates of this calibration level and then add the next
level replicates.

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 About Data Handling
Process / Review RecalcList

When all the calibration files have been entered, Save the RecalcList and close
the Automation File Editor.

Process / Review RecalcList

Click on the MS Data Review icon in the Toolbar to open MS Data Review.
Cancel the Select Plot dialog and pull down the Quantitation menu. Select
Process/Review RecalcList.

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In the Method File line, Browse for the method file you wish to calibrate, then
Browse for the RecalcList. Click on the Process button to execute the
recalculation. As the recalculation proceeds, a line by line report of progress is
generated, including any instances of missing or miscalculated compounds. If
the recalculation is completed without any faults, you can proceed to view the
calibration curves.

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 About Data Handling
Process / Review RecalcList

If some compounds were missed or miscalculated, double click on the line in the
RecalcList in which the problem occurred to bring up a results table for that file.
Then, double click on the line for the missing or miscalculated compound. The
Compound Integration window which is displayed will show the peak profile and
the results of the integration, indicating what aspect of the peak quantitation
failed.

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Modifying a Method
Click on the Edit Method button to access the Quantitation Parameters for the
compound and modify the method so that the peak is properly quantitated. Click
on the Integrate button to recalculate the results and note whether the
compound is now properly processed. Click on the Done button to return to the
results list.
When all the compounds at all calibration levels are properly detected, click on
the Done button and save the changes to the method. Reprocess the RecalcList
with the modified method to generate the Calibration curves. When the
recalculation is complete, click on the Edit button to open the method in Method
Builder, and double click on the Compound Table. Finally, click on the View
Curves button to review the calibration curves for each compound.

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 About Data Handling
Modifying a Method

You may modify the Curve Fit Type, the Origin Treatment, and the Regression
Weighting Formula to optimize the fit of the data points to the calibration curve.
You may also examine the individual replicate points. When you are satisfied
with the results, you may print the curves.

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