Category Archives: Method Development

New Confirmation Setting in TraceFinder 4.0 and above

TraceFinder has always had a plethora of criteria that is available to assist in the review of data quality. Below is a picture of the Chromatographic Suitability panel that can be set for each peak individually or as a global setting. This flagging criteria has existed since Xcalibur’s creation in 1994. However, unlike Xcalibur, TraceFinder will report out the actual measured value of each set criteria versus the simple PASS or FAIL. These values can be visualized in the data review summary flags or utilized in creating reports.

However, on each type of confirming criteria (Ion Ratios, Isotope Patterns, Library Searches, etc..) there exist the ability to turn them on or off for the method. Added in the TF 4.0 release is the ability to enable the Required option. Setting this option allows the users to control for peaks that only pass the set criteria. All peaks that have the Required option turned on for one or many confirming criteria and have a failed value for the Required option will not be intergrated. Thus, it will be as if the peak was not found in the sample.

This is an optional feature and is turned off by default when the compounds/peaks are created in the Master Method Template.


Tip for Optimizing the Speed of Processing Data for TraceFinder 4.0 and later

TraceFinder 4.0 introduced the ability to perform target analysis on peptide sequences. Given that these chemical analysis methods can be very long (i.e. 3hours for a single sample) and have drastically shifting retention times between samples, TF 4.0 introduced the Extraction Time Window as a peak detection setting.

This new setting allows the user to set a window around where the peak might elute. The default setting is 3 minutes, which means +/- 1.5 minutes from the expected retention time.

This setting may be a bit wide for stable chromatography. A typical setting for the Extraction Time Window is 1 minute or +/- 30 seconds from the expected retention time. This setting can be changed in the Master Method Template or the Local Method.  This new setting can then be pushed to all the compounds with two mouse clicks or can be optimized for each target peak, clicking the Three Lined Button in the right coner of the settings page and selecting “Apply current extraction window settings to all peaks in the method

Since the amount of data being extracted for the targeted peak is reduced, the amount of time per peak analysis is reduced. This setting is separate from the Peak Detection and View Width Window under the Retention Times Tab.


Peak Extraction Window

Retention Time Reference Compounds Used For Correcting Chromtographic Drift

TraceFinder has an option to allow the user to set a reference compound to assist in corrections of retention time drift.

The feature is the same implementation that has been a part of the Xcalibur feature set for over 20 years and is well characterized. The user simply selects a checkbox in the RT Reference compound column of the Method – Identification tab. This sets the compound for use in linking other compounds to its found RT.

To set a compound to use a RT reference compound, simply select a RT Reference from a dropdown  selector in the same definition grid.

RT reference

The typical use of this feature is to set your Internal Standards as RT Reference Compounds and then link the nearby eluting components to the Reference Compound. Also, the IS retention time peak detection window is set a little larger than the target components to allow for detection if a large RT drift occurs.

When the RT reference is detected the difference, whether + or – in time are adjusted in the connected compounds. This new RT is specific to each compound in each sample. Therefore it is not reflected as a change in the method because the method is set as a batch level setting. The compound using the RT reference must have an original expected retention time to be adjusted, it cannot be left as zero for this feature to work.

How to use the Threshold Sample in Comparative View

To utilize comparative view to its fullest, one must set 3 three properties.

  • Method-Limits-Threshold setting – this setting allows the user to set the height of the horizontal threshold bar in data review. There are two settings.
    1. Threshold – a fixed peak height amount
    2. % of Threshold the Sample – dynamic threshold according the peak height of the specified Threshold sample, as described below.

Method Threshold Setting

  • Batch view-samples-groups column – this setting assigns samples to groups that are displayed horizontally in the comparative view peaks panel. A unique alpha numeric symbol is used to assign a sample to a group. Each sample with the same symbol is a member of that group. The samples are shown side by side in the peak review panel and are ordered left to right by descending order in the sample grid above.Batchview Threshold Setting
  • Batch view-threshold samples – the setting allows the user to assign a threshold sample for each group. Any member of the group can be set as the threshold sample. However, only one sample can be utilized at a time in the group. If you want one particular sample to be used as the threshold across the batch make sure it is a member of all the group. For example if you used numbers for the group name and you have 10 groups, the entry for this sample in the groups column would be 1,2,3,4,5,6,7,8,9,10.Threshold sample selection

The settings in the examples above allow for the display shown below.

The tall red vertical bar marks the expected retention time and the smaller vertical bars are the edges of the retention time window. Both set in the method parameters for each compound. This allows you to see the drift of chromatography within the sample group. The horizontal red line is the threshold as set in the method as a fixed amount or relative percentage of the threshold sample, as set in the method.

Comparative View in Data Review

A point to note, there were a few instances where everything was setup properly but nothing displayed in the Comparative view. It was found that some older  video drivers not comply with technologies Microsoft used to render the images in this complex grid. However, the issue was fixed in all cases by updating to the latest video drivers for the PC.

Ion Ratios – How the range is calculated in the Methods, with added twist in TF 3.3

TraceFinder has 4 functions for ion range calculations.

Ion Range

1) Manual – This functions as the name implies, it allows the user to define a targeted ratio by manually entering it in the ratios section of the detection tab of the confirming ions of a compound.

2) Average – This functions allows the data to drive the targeted ratio of each confirming peak by averaging the detected peaks for each confirming ion in each component from the calibration curve and applying the average as the target ratio. This is useful in many clinical experiments where response may be variable across sample sets.

3) Weighted Average – Performs the same function as Average with the exception that it weights the bottom end of the curve in the calculation of the target amount. This is useful in assays where the expected amounts in the unknown sample types are typically on the lower end of the calibration range.

4) Level – This selection allows the user to select a level from the calibration curve as the target ratio reference. In some labs the SOP’s  require that a specific calibration point be compared for the batch of samples.

The selection of 2-4 are widely used in the applied markets. This relieves the use of having to perform the calculations manually outside of the application and then hand entering the data.

Also to note is that if you are working in a batch of samples such as a verification samples set. You may use settings 2-4 to calculate the values then changes the setting to manual. This action keeps the calculated value in the target field. you can then update the Master Method template and use these ratios for all validated batches to follow.

TraceFinder 3.3 has the benefit of being able to store the ion ratio criteria in the Compound Database so that you may perform quick holistic edits or use the same criteria in different assays without recalculating the values and manually entering them. As with the methods themselves, each ratio has properties that can be set as:

  • Window Type – How to calculate the width of the range around the target ratio
  • Ratio Window – The amount of the range
  • Coelution Value – The amount in time that the peak apex of the confirming ion can be different from the quan peak.


The file hyperlinked below is an example CSV file template for a compound database for TraceFinder 3.3. This file will allow you to record settings from other methods. If you are transferring from one instrument platform to another, this enables you to quickly create the same method in TraceFinder.

Click here to download the ZIP file -> Default

As a reminder TF can group in the CDB and the Method up to 6 components as individual integrated quantitative peaks under the compound name. This allows the peaks to be viewed and manually manipulated individually in data review but the responses summed as the total response of the compound. Each of the 6 quantitative peaks may have up to 10 confirming peaks with individual ratios and ranges.  (A key note is that the Quantitative peaks need NOT be coeluting. (ie.. Chiral compound analysis)) This feature is mostly when using GC EI experiments or LC experiments with multiply charged components.

TraceFinder 3.2 – Method Setup Tweak – Threshold Setting

There have been a few questions on the issue of peaks not being integrated, while performing quantitation, although the signal is strong. A reason could be that when a compound is created in the method, it has a related compound database entry for a threshold setting. This setting is used in targeted screening but is not editable in quantitation mode.

The setting has a default value of 5000 for calculated peak area. This setting does have a global override in the Local and Master method  Processing section. The default value is unchecked and blank. This means the default threshold is 5000 for all peaks. If you are having an issue where the peak is not integrated but has a strong signal, then change the setting by checking the box and entering a new value such as 0 or 500. The data will need to be reprocessed as this value is not automatically applied.

Threshold 1


TraceFinder 3.2 – New Quick Acquisition for Method Development

In previous versions of TraceFinder, there existed the Development Batch under the Method Development Mode of the application.

TraceFinder 3.2 has been replaced  with a new Quick Acquisition button in the toolbar and in the Tools menu items.

This allows the user to acquire a sequence of files and open them in either Qual Browser or FreeStyle, from any view or mode.

The main benefit is that the user can perform method development or simply acquire a file to test the system. This is done without having to utilize batch processing to get an quick answer to the question.

Video player has changed. If the picture appears blurry, in the top right hand corner of the player you can enable HD to play back high resolution video.

TraceFinder 3.2 – Intelligent Sequencing as a Standard Feature

TraceFinder 3.2 will include Intelligent Sequencing as part of the standard features set.

Previously, this was an additional add-on to the TF applications, with a separate license.

Now Intelligent Sequencing will be able to be configured in the Application Configuration section of TF, as shown below.

If you are not familiar with Intelligent Sequencing, it is the capability to perform actions on the sequence list of samples from real-time analysis of the flagging system.

The uses of this feature are numerous, the main benefit is that you have a safeguard for your samples.

– If you have a QC sample that is out of tolerance, you can stop the acquisition of following samples.

– If you have carryover in your blank, the system can automatically introduce additional blanks until it is clear or stop the sequence after so many blanks have been injected.


App Configuration



Xcalibur Quan Method to TraceFinder Methods for Quan Conversion

Below is a quick and simple way to move from Xcalibur based quan methods to TraceFinder.

If the video is blurry please click the cog wheel at the bottom of the panel and increase the video display resolution.


Isotope Pattern Scoring in TF 3.1 – Answer to “Ask A Guru” Question

Below is an excerpt from the online help in TF 3.1.

A few questions have come up on how we calculate the isotope pattern score and visualization.

Here is the answer directly from the online documentation. Remember we do have comprehensive manuals and help under the Help Menu.

Link to PDF file: isotope scoring

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