Category Archives: Method Development

Intelligent Sequencing – Step One – Setting up the method

Intelligent Sequencing is not a new concept. In the late 90’s Thermo had an application called ToxLab which used the concept of flagging data and altering the sequence of the set of samples based on failure criteria.

This allows for insertion of blanks, reinjection of sample or a combination according to criteria that a chemist would normal use to look at his data the next day to determine, which if any samples had to be rerun.

Intel Seq just does this on the fly.

It allows the user to have confidence that the data will be handled in the prescribed manner using the existing on the fly criteria that has existed in TraceFinder for a number of years.

By using this feature the chemist can cut back time on reinjections and sometimes on sample prep since the sample wouldn’t have evaporated. Also it will not have gone beyond the sample stability time threshold before reinjection occurs.

The first part of the operation is setting flagging criteria as you have done previously.

The next is setting of the method criteria for Intel Sequencing.

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

The Master Method – A trinity of information

We will start a short series on the Master Method. So here we go.

Think of the Master Method as the supply truck that comes everytime you want to run an assay. It contains what you need to get things done. It’s alittle bulky and can be somewhat intimidating the first time you see it. Since it controls alot of the available power TF gives you, it has lots that can be,but doenst have to be configured and used.

For example, if a value is set to zero in the QAQC section it isnt turned on for use by the flagging system.

But lets start off with the basics.

The Master Method is a repository for the three things needed to gather and process a data file.

  1. The instrument method
  2. The peak processing parameters
  3. The selected reports to be produced.

With these three things we can submit a batch of samples and acquire, process and produce reports on the fly, after every sample. (but you need a sample list too)

The instrument method is a copy of a stored instrument method somewhere on your hard disk. It is associated with the processing method and can be changed according your your needs. But you always have the ability to go back to your orginal instrument method and copy over any changes. It’s a safety net.

It also insures that you only have to pick the Master Method when making a batch of samples. This keeps a batch consistant, run after run and technician to technician.

The processing parameters are set up for each peak and compound grouping. Once set up again it keeps things consistant.

Thirdly, is the report section. This seems to be a bit of confusion at times. By selecting which reports and what format you want them produced in, ahead of time. The data is always produced in the same way without missing a page. It’s also nessary for the reporting on the fly feature.

If you want to create reports after each injection, simply check the box on batch submission that says “Create Reports”. Then however you selected the report to be produced and one or multiple formats, it make them once the sample has been acquired and automatically processed. Except for Batch level reports, which are produced at the end of the batch sequence.

Follow the video below to see the process.

The video is from the soon to be release TF 3.0 but the workflow is the same.

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

Need to just enter some compound info for the method… Ok here’s a solution

So at times we’ve had the request that chemist just want to enter in some compound data without having a rawfile to extract the spectra from.

And though TF gives you multiple ways of creating methods, some feel they just want to be able to add a compound and input their data by hand.

Though many workflows support doing this in using a CSV file and creating a method from importing that csv file into the CDS, sometimes that just may be too much.

So here is couple of CDS files that you can have on hand just to pull over and fill in place holder information.

One is for XIC/SIM type of experiments and the other is for SRM type of experiments.

https://dl.dropbox.com/u/13860428/Blank%20XIC%20Compound%20CDS.csv

https://dl.dropbox.com/u/13860428/Blank%20SRM%20Compound%20CDS.csv

Making changes in to the multiple peaks in a compound (Answer to a “Ask A Guru” question)

There are times that a chemist may make a method from a PMD file or from CDS, and things need to be changed.

An istance would be swapping an existing quan peak with its confirming peak, or removing a confirming peak.

These changes while seemingly trivial can be a hassel if you have to do many of them. So the TF team has given these options as right click contect menus to make it easier to perfrom these functions.

Watch the video below to get an idea of how to use these tools.

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

Matrix Spike and Matrix Spike Duplicate… Understanding the how it works.(Answer to a “Ask a Guru” question)

A question from the Ask a Guru was how to use MS/MSD samples and reports, well read below.

In the Environmental realm there is an experiment for a Matrix Spike and a duplicate, when compared to an unknown sample.

This allows for the chemist to calcualte a recovery of of the compounds contained in the unknown sample.

To set up the experiment the chemist needs three sample types.

  1. Matrix Spike – with compounds spiked into it at a known level
  2. Matrix Spike Duplicate –  with compounds spiked into it at the same level as the Matrix Spike
  3. Unknown – a sample with unknown amounts

Under QAQC tab of the Master/Local Method the concetrations of the spikes compounds must be entered in the grid for the MS and MSD tab.

In the Batch View the samples to be grouped together and used to report the Recovery must have the same SAMPLE ID. There must be one of each SAMPLE TYPE, but each one must have the sample SAMPLE  ID text in their individual grid cell.

When the batch is processed the data for recovery will be calcuated and a MS/MSD Report can be generated that displays the information about this experiment.

See the picture below of the report in Report View.

Thanks to Gail Harrison for her suggestion on a blog post.

Need To Look At Files With Data Dependant Experiments?

Though TraceFinder is primarily a quantitation application, it does have some provisions for unknown identification.

Mainly used in Single Quad or Ion Trap Analysis, the qualitative processing allows for scanning files along a TIC for peaks of interest and then eliminating the known targets and performing library searches against the unknown peaks. Also, TF gives the ability to look at the peaks that generate data dependant scans(DDS). It will sum the spectra and compare to a NIST style library.

To utilize this feature you must have two things. One a Method template, with the parameters set for Qualitative processing and DDS enabled, and a Sample Type of either and Unknown/TIC or Unknown Qual.

If you are performing Quantitative processing those will be perfromed first and Qualitative will occurs on the sample after Quan results are created.

If you have Qual resuts in data review you will have a green button to select the QualView.

Follow the the Video in setting up a Method Template and then using it in a Batch to review qualitative results.

If the video is blurry select the cog wheel in the lower right of the video window and adjust the resolution setting.

Add A Confirming Ion From A Compound With A Separate Mass Filter

At times compounds must be confirmed from another compound using ion ratio.

Here’s how to set it up with SRM and SIM type of experiments.

Make it easy on yourself… Update in one place

In the Master Method you can review settings for your Triple Quad mass specs, such as Retention time, window of detection, collision energy…..

You can also make adjustments in the Master or Local Method to your Retention Time. Either by manual entry or by using several ways of performing automatic adjustments.

The issue comes in that when you make these adjustments in processing part of the method, the instrument method doesn’t know about them. So if you are using TSRM  you have to update in two places.

The Acquisition List is updated with the changes to windows and Retention times and can produce a file that can linked or exported out for adoption by the instrument method.

This allows you to edit in one place and by using the Auto TSRM update check box on the Acquisition section, You’ve just now reduced the typing and file coordination, that has to be done.

If the video is blurry select the cog wheel in the lower right of the video window and adjust the resolution setting.

Changes to My Method… Alot of them… Really?

So as a chemist sometimes it’s hard to believe that I didn’t make the perfect method.  Well, not really. Things change, cloumns go bad, my boss changed the SOP.

So considering the Master Method is my template used for creating batches with the same compounds and parameters, and it can hold up to 3000 compounds, which can have up to 66 components each, I don’t want to have to manually adjust each component.

In each section of the Compound detection tab there is a right click option that allows for copying of those settings to each component in the particular compound or across the entire method.

We hope this saves you time and lets you hit the weekend sooner so your not rerunning samples on a friday night.

If the video is blurry select the cog wheel in the lower right of the video window and adjust the resolution setting.

Edit Instrument Methods in Master Method

In the Master Method, a user assigned an instrument method that resides in the c:/Xcalibur/Methods folder. This method is then copied into the Master Method folder when the Master Method is created. Thsi allows the Xcalibur copy to remain as a safe back up.

Two things can be done, you can open the Xcalibur method up and make changes and do method development in xcalibur, and once satified, update the Master method from the altered Xcalibur Method. Alternatively, you can edit the Master Method copy of the instrument method and once satified update the c:/Xcalibur/Methods folder copy of the instrument method so that new Master Methods created with that instrument method will inherit the changes you made.

If the video is blurry select the cog wheel in the lower right of the video window and adjust the resolution setting.