MultiTrace Tutorial: Evaluating a sample

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MultiTrace 3: Tutorial

In the second lesson you will learn how to open and evaluate a sample. You will also understand the concept of MultiTrace's calculation.

Contents

Opening a sample

image:Tutorial-OpenSampleButton.png

Please click on the open sample button in the upper left corner.

The open sample dialog will pop up, which looks quite similar to the open reference dialog. The open sample dialog is split in three parts. On the left hand side you find the file explorer, the right hand side is split in an upper part which shows you all runs (GC/MS data files) present in the current sequence. The lower part shows a list of result files available for a certain sample file. At the moment no data file is selected and no old results are available, so the lower list is empty.

Please double-click the 31943_D1_E1184.D sample file in the upper list. Alternatively, you may select this entry with a left click and then click the open from scratch or open button in the lower right corner.

The sample dialog only shows data files acquired with the same method (here MS3) as the opened reference. If data files are available that have been acquired with other methods, they are hidden and do not appear in the upper list. This faciltates and speeds up the selection of appropriate samples. Thus, longer sequences are reduced to partial sequences of the same acquisition method.

image:Tutorial-OpenSample.png

Main view explained

The main view now displays all data relevant to the opened sample, its associated reference and method.

If not already the case, please select the first entry, Alachlor.

image:Tutorial-MainViewSample.png

Please note that MultiTrace shows the sample, its integral area and integral values in blue. MultiTrace automatically integrated the sample and calculated the possible concentration of the parameter for both ions. The values are displayed in the top of the graphs as well as in the table.

Auto-zoom

You can use all zoom techniques described in lesson 1, e.g. mouse zoom, mouse cut, or time shift. However, MultiTrace automatically zooms the blue trace appropriately and in most cases, no further zoom should be necessary.

The blue trace (sample) and red trace (reference) usually do not have the same intensity axis. The intensity axis shown in MultiTrace is always valid for the red reference trace. The blue sample trace is usually zoomed. Please note the blue number (like "9.6 x") in the lower left corner of each ion trace graph. This number is the factor with which the blue trace is zoomed compared to the red trace.

You can toggle the auto-zoom on and off by clicking the auto-zoom button or by hitting the "z" key on your keyboard. If there es no blue factor in the lower left corner, the intensity axis is valid for blue and red and both traces are shown in their actual intensities. If there is a blue factor in the lower left corner, the blue trace is enlarged by this factor.

By default, MultiTrace zooms the highest peak of the blue trace to a 40% level of the maximum intensity. This level can be customised in the options dialog.

Assigning a comment

We determine that ion trace m/z=160 shows no relevant peak. The parameter is not present in the sample. The ion trace m/=188 shows an interfering peak. We can decide to just assign a negative evaluation result to this parameter or to additionally assign a comment stating the interference for the report and future assesment.

There are three methods of assigning a comment:

  • hot key comments: Hit the key F1..F4 to assign a comment to the first ion, F5..F8 for the second ion, or F9..F12 for the third ion. There are only four different hot key comments possible and they are identical for each ion. The comments' text can be edited in the configuration file (see Reference Manual: Comments). This method is the fastest way to set a frequently used comment, and it is easy for a group of co-workers to set identical comments, since the texts are defined globally.
  • rotational comments: Hit the key "o" once, twice or more often to cycle through a set of comment texts and assign them to the first ion. The key "p" applies to the second ion and "ΓΌ" (German keyboard) or "[" (English keyboard) to the third ion. Use "Shift" together with the key to reverse the cycle order. This is the old method of MultiTrace 2 and has the advantage to be able to define more than four comments, in fact as many as you need. It has the disadvantage that comments late in the cycle order need several key strokes. Rotational and hot key comments are independent from each other and can be used in parallel.
  • free edit: Just hit the hot key "c" to open the comments dialog and enter whatever comments you need for each ion. Alternatively, you may open this dialog by clicking on the area where the comment text will appear in the graph title bar.

Online help

MultiTrace has many hot keys to speed up the work flow. Most features you can also activate by mouse, but like with the comments in the previous step, hot keys are faster.

As new user you might want to stick to the mouse, but we recommend to memorise and use hot keys. In the upper right corner of the main view is a "?"-button offering online help and displaying a table of all available hot keys. Just have a short look!

You may also access this Wiki tutorial and manual through the online help page by clicking the hyperlink in the upper right corner.

Assigning an evaluation result

image:Tutorial-EvaluationButtons2.png

Returning to the Alachlor evaluation, we decide to mark this parameter as negative. To do so, we hit the hot key "-" (minus) or click on the negative evaluation button on top of the ion trace graphs. You may hover the mouse over the evaluation buttons to see their respective hot keys.

MultiTrace will add the negative result in the table's rightmost column "Eva". The color indicates a proper result has been entered, the "<" indicates the negative result. MultiTrace automatically moves to the next parameter and auto-integrates it. We can easily determine that the second parameter is negative, too. Just hit the "-" (minus) key again.

MultiTrace automatically selecting the next parameter usually makes sense and speeds up evaluation. You should only assign an evaluation result when you are done with integrating and deciding. However, it is possible to turn this feature off (see MultiTrace: Options).

Calculation

The method definition file gives the concentrations of all parameters present in the reference mixture in terms of the primary unit and it defines which unit this is. In our demo file, the primary concentration is "ng/ml". The concentration of Brompropylat in primary units is 200.50 ng/ml as shown in the table's "c(Ref)" column. You will later learn how to define your own methods and their units, concentrations, how many digits to use and so on.

MultiTrace automatically calculates by rule of proportion what concentration each quantifying ion would lead to and displays the result in the table and on top of the ion trace graph. It uses the same unit as the primary unit of the reference concentration. The result is shown in the column "c (ng/ml)".

Dilution factor: MultiTrace can take a dilution factor into account, if the sample is diluted in relation to the reference concentration. This factor is mulitplied into the primary concentration result.

Weight factor: MultiTrace converts the primary concentration to a secondary concentration with a different unit (here "mg/kg") by dividing through a so-called weight factor. This factor has to take all aspects into account that are required to convert one unit into the other. You are free to define this for yourself.

Usually, the secondary concentration relates to the concentration of a parameter in the material sample itself, while the primary concentration relates to the concentration of a parameter in the analytical solution injected into the GC/MS system.

Usually, dilution factor and weight factor are taken automatically by MultiTrace from the raw data file supplement data fields (like "miscellaneous info" or "sample name" of the ChemStation sequence table) or from the raw data filename. For example, the demo file 31943_D1_E1184.D gives the sample ID number (barcode number) 31943, the dilution factor 1 and the weight factor 1184. If you name your file in this manner, MultiTrace will automatically extract the values accordingly. However, there are several other ways to make MultiTrace take these values from the raw data files and you should discuss with our support which way is the best for your laboratory workflow. We will support you to find a solution with as little change in established workflow and maximum comfort.

Re-integrating the sample

You may re-integrate the sample exactly as you do with the reference. If the blue integral is visible, you will re-integrate the sample; if the red integral is visible, you will re-integrate the reference. You may switch between reference and sample by clicking on the little red or blue square in the upper right corner of each graph, directly right of the concentration value.

Alternatively, you may simply hit the space bar to toggle between the red und blue integral.

Possible evaluation results

You have several options of possible evaluation results:

  • positive (compound found), green color
  • questionable (uncertain result, to be discussed), orange color
  • negative (not found), green color
  • n.a. (not analysable), green color
  • ignore, gray color
  • unknown (not yet evaluated; default setting of new samples), white color

You may internally decide, how to use these options in your laboratory.

Our recommendations are to use "questionable", which is marked in orange, only for those parameters you want someone else to become aware of. Samples with questionable parameters are highlighted in the open sample dialog, so as to make supervisors or co-workers aware of these samples, that clearly appear as not ready. Instead, use "n.a." to indicate you finished up with a parameter, but could not determine a clear positive or negative result.

We further recommend to use "ignore", which is marked in gray, only for those parameters actually ignored, i.e. not evaluated at all., e.g. because it is missing in the reference, for quality control, an internal standard, not ordered by the customer or not part of the current test method.

You can select evaluated parameters and change the evaluation at any time. You can use the evaluation buttons or type the associated hot key. However, re-integration is only enabled, if the evaluation result is "unknown". Press "0" (zero) or click "unknown" to re-open a parameter for integration. This feature is meant to minimise unintentional changes. However, you can deactivate the blocking in the options dialog.

Filtering the table by results

On top of the table are small buttons that enable filtering the list of parameters by their respective evaluation results. This is particularly sensible for methods with more parameters than visible rows. You may change the filter by clicking the appropriate button or by using the cursor arrow keys left and right.

  • pos: positive
  • que: questionable
  • neg: negative
  • n.a.: not analysable
  • ign: ignore
  • eval: evaluated, i.e. pos, que, neg, n.a. (excluding ign or unk)
  • exp: matching export criteria (printing or LIMS)
  • thr: positive or above threshold
  • unk: unknown (not yet evaluated, e.g. forgotten)
  • all: all parameters

The "export" filter is only applicable, when extended database or LIMS features are activated. This usually requires customisation of MultiTrace. Please ignore this filter as beginner.

Directly after opening a sample, MultiTrace automatically selects the filter "all". You do not have to reset the filter manually, if you filter, print, and open the next sample.

Saving and closing the sample

Auto-save

With MultiTrace you will not loose work or results. You may shutdown MultiTrace at any time and the current state of evaluation including integrals, comments and results will be saved automatically. You can shutdown MultiTrace by hitting the key "Esc" in the upper left corner of your keypoard, by clicking the cross in the upper right corner of the window or by hitting Alt-F4 to close the main form.

  • You may simple click on open sample and open the next sample and MultiTrace will automatically save the previous sample. This is the default workflow.
  • You may simple click on open new sequence and start with the next sequence and MultiTrace will automatically save the previous sample, close the references and start from scratch. This is the default workflow when having finished a sequence.
  • You may click the close sample button in order to close the sample and return to the reference. Usually, this is not necessary. If you are administrator and want to edit the method, you might want to close a sample and work with the reference only.
  • If you want to close a sample and intentionally discard all changes and not save the results, you can hold down the Ctrl-key and click the close sample button. MultiTrace will then discard all changes and close the sample without further ado.
  • If you insist on saving a result file while working with a sample, you may click the save sample button. However, generally there should be no reason to do so.

MultiTrace saves the results in a text file in Windows configuration file format (*.ini) inside the raw data file folder. The files begin with "MT3-" to indicate they are created by MultiTrace, and also include a date-time stamp and the user name (login name of Windows sesssion). You may open the result ini-files in a text editor should the need arise or if you want to process them with a third-party program.

MultiTrace will hold an unlimited number of result files for each sample. Thus, the whole history of evaluation is available and re-evaluating a sample does not discard previous results. Even in the case several co-workers work (accidentally or intentionally) on the same sample, the result files will not collide and can be easily assigned to each person.

Continuing the tutorial...

Please click on the open sample button. You are back to the open sample dialog. Please select the sample by a single left click on its row. The lower result files table will now display one line -- your results which have just been saved automatically as you clicked on open sample. The result files table shows which evaluations occur how often, the time-stamp and user name tell you by whom and when this result file has been generated and the color in the righmost column indicates whether there are still questionable or unknowns in the parameter list.

  • the color code is orange if unknowns or questionables are present, indicating "not ready"
  • the color code is green if no unknown and no questionables occur

Please select the result file by double-clicking it or by selecting it with one click and then click open. MultiTrace will open the associated reference file(s), the sample raw data and the result file. MultiTrace will select the parameter you had selected when the result file was saved, so you can continue your work exactly where you left.

Now please play around with the possible evaluations and repeatedly re-open the sample dialog. Watch the number of result files grow and check the orange/green code depending on what kind of evaluations you choose.

Usually, you only open a new sample if you are done with the previous sample. Thus, generally only one or only a few result files per sample are generated.

Open from scratch

If you want to start over the evaluation of a sample from scratch, just click the open from scratch button. MultiTrace will the open the sample raw data file and display the sample as if no one had worked with it before. Of course, when opening a sample from scratch, a reference file of the same acquisition method has already to be open.

If no previous result files exist, open and open from scratch are identical.

Please note that you will not loose previous work, if you or a co-worker open a sample from scratch that had been evaluated before. Instead, just one more result file is saved and when opening the sample again you will have the choice between all existing result files.

image:Tutorial-OpenSampleResults.png

Marking a sample as ready

image:Tutorial-MarkReady.png

When all parameters have been evaluated, i.e. when no parameter is unkown, a little square appears right of the print buttons. This button can be used to explicitly mark the sample as "ready". When clicking the mark ready button, the sample is immediately saved.

The square symbol is shown in the open sample dialog in the color code column. It can be both on orange and green codes. It shows you, whether the operator had decided to explicitly mark this sample as ready. Whether you use this feature or not, is up to you. There is no internal requirement to do so, but it may facilitate your laboratory workflow and team communication, particularly if more than one person is required to finish a sample's evaluation.

Enhanced feature: Result file ID -- MultiTrace can be configured to assign a result file ID to each result file and to store this ID in a database. The result file ID can be printed as barcode number on reports and later on this result file can be opened by simply entering or scanning the barcode number. MultiTrace will then open the reference files, sample file and result file from the appropriate instrument or network folder. This is the most rapid way to open old evaluations when a printed report is given. Even months or years later you will be able to simply scan the barcode and have all data ready to peruse. For details about this feature please read Reference Manual: Result file ID.

The footer of pages printed by MultiTrace will then optionally show the additional barcode number in the center:

Enhanced feature: Eva -- Eva 2.x is a software employed by the Eurofins group to access, select and qualify all results related to a certain sample ID, e.g. it displays a collage of results obtained by GC/MS, LC/MS, GC-FPD, GC-ECD and many more analytical techniques. MultiTrace's result file ID feature allows to click on a result value inside Eva 2.x and remotely control MultiTrace to open the associated result file (including references and sample) and even select the appropriate parameter, so that you can see the ion trace and integral that gave rise to this value.


Continue with the next lesson of this tutorial: MultiTrace Tutorial: Printing results

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