MultiTrace Tutorial

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MultiTrace 3: Tutorial

We believe that MultiTrace is one of the most convenient software systems to do GC/MS-SIM analytics with and we hope you will become an enthusiastic user as well. We are confident that with these step-by-step tutorials you will become an expert MultiTrace user in a very short time.

We offer all customers full support by email, phone and remote access using TeamViewer. If you have got any problems, inquiries or suggestions on how to further improve MultiTrace, please do not hesitate to contact us. We will do our best to solve all issues reported and to implement new features free of charge we consider to be of interest for the majority of users.

If you have very special requirements and wishes for new features, we will gladly give you a free quotation for a customised, individual version that solves your institute's or company's challenges. This particularly applies to database access, LIMS import and export, automation, barcode usage and individual report requirements.

Contents

Introduction

This tutorial uses a step-by-step approach to teach the fundamental aspects of MultiTrace. If you follow each step, you will swiftly and easily learn how to use all important features. Please refrain from using your own data files at this moment; the tutorial data files are selected to offer the best and most rapid access to all important features. Later on in this tutorial you will learn how to analyse your own data files.

Please note that the tutorial does not cover all features and options of MultiTrace. A complete explanation of all details of each dialog as well as all hidden features for database acces and customisation can be found in the MultiTrace Reference Manual.

By the way, as mentioned in the installation guide, we recommend to use a large monitor with a high resolution. 1920x1200 on a 24" screen would be perfect.

Starting MultiTrace

Please ensure the dongle is connected to your computer or available through your local network. Start MultiTrace by double-clicking the executable or by using the shortcut on your desktop or taskbar.

MultiTrace will start with the main view as shown below.

image:Tutorial-MainViewEmpty.png

Fundamental principles

MultiTrace is made for analyses following the basic concept of directly comparing a reference GC/MS run with a sample run measured under identical conditions, usually closely after each other.

  • The reference run contains all analytes of interest in known concentrations.
  • The sample run may or may not contain the analytes of interest. The concentrations, if present at all, are unknown and to be determined by the analysis.
  • The reference run is used as external standard and comparing the integerals of a certain analyte in the reference with that of the sample allows to estimate the amount of analyte contained in the sample.
  • All GC/MS peaks and their integrals relate to ion traces extracted from the measured mass spectra. The raw data may be single-ion monitoring data (SIM) or full scan data, the visualised data are always extracted ion traces. It is possible to use up to three ions per analyte. All ions are quantifiers and qualifiers at the same time.

In MultiTrace this procedure is carried out in the following order:

  • You open a reference file and integrate all analytes.
  • You open a sample file, integrate all analytes, and print or export the results.
  • You open further sample files to be compared with the same reference file.

Integrity of raw data

MultiTrace reads Agilent ChemStation data files (../SAMPLE.D/DATA.MS), but does never write or modify such data. Your data files are never touched in any way. MultiTrace does not even have any routines implemented to write or modify raw data.

MultiTrace does not read or process any other files present in the sample folder nor does it delete or modify any of these files. You may store further data in the sample folder without MultiTrace noticing or interacting with them in any way.

MultiTrace does not collide with other evaluation software nor with Agilent ChemStation itself. You may evaluate data with Agilent ChemStation or any other analytical software without problems. This allows for easy quality control and smooth transition from your old evaluation system to MultiTrace.

MultiTrace writes its own result files as human-readable text files (configuration ini file format) into the sample folder. The filenames clearly state their application association, user name, and date-time stamp. These result files contain all evaluation-relevant data and calculated results generated by working with MultiTrace. If you no longer need these data, you may delete the result ini files without disturbing MultiTrace (however, you will loose the results).

Opening a reference

Please place your mouse pointer over the icons in the upper left corner. Note how a bubble hint pops up when you hover the mouse for some seconds. This principle works throughout MultiTrace and provides you with hints about the user interface elements.

Please click on the "open new sequence" button in upper left corner.

The open reference dialog will pop up. The dialog is split in three parts. On the left hand side it shows a file explorer, with which you can select drive and folder of the sequence to work with. You can also access networks drives exactly as you would do with the Windows Explorer. For the moment, please don't change anything, but use the demo files.

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 all references currently selected. At the moment, this list is empty.

Please double-click the REF.D line to add this data file to the list of references in the bottom half. Please note that MultiTrace displays with which method the run has been acquired (MS3) and will select the appropriate method definition file automatically.

In this tutorial we will only use one reference data file, so please click the open button in the lower right corner. MultiTrace will read the selected reference data file and the associated method definition file, then close the open reference dialog and return to the main view.

image:Tutorial-OpenReference.png

Main view explained

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

image:Tutorial-MainViewReference1.png

Result table: On the left hand side is a table with all analytes of the method. A method is a list of parameters and contains each parameter's name, gives the ions to be used for quantitation, the time window to pick the peak from, and the known standard concentration in the reference mixture. A method also contains some header data such as which units (like "ng/ml") to be used. You will learn how to define and edit your own methods in a later lesson. At the moment just note that MuliTrace recognised with which method ChemStation had acquired the GC/MS run and automatically opened the appropriate method.

Further note, that MultiTrace already automatically selected the first parameter of the method, here Alachlor, and extracted the associated ions m/z=160 and m/z=188. It knows that its concentration in the reference mixture is 200.50 ng/ml.

Ion trace graphs: On the right hand side there are three graphs from which two graphs show the extracted ion profiles of the m/z=160 and m/z=188. The third graph on the right is empty because this parameter uses only two ions.

The method definition told MultiTrace which time range to display and from which time range to pick the largest peak. MultiTrace already integrated the peaks and displays the integral both in percentage and absolute counts in the upper left corner of each graph. MultiTrace repeats the reference concentration in the upper right corner of each graph.

Please note that MultiTrace always display the reference in red. The extracted ion profile, the integral area and the reference concentration are shown in red.

Navigating through the parameter table

Please turn the mouse wheel up and down. Note, how you scroll through the parameter list of the method table. MultiTrace instantaneously extracts the relevant ion traces, picks the peaks and integrates them.

Alternatively, you may use the cursor up and down arrows an your keyboard. Most of MultiTrace's operations can be done using hot keys.

Alternatively, you may select a parameter by clicking with the mouse anywhere on the desired line of the method table.

Integration

In many cases, MultiTrace's automatic integration will be fine. However, sometimes you might want to re-integrate a peak manually. MultiTrace's integration feature is very intuitive and easy to use. You have two primary options to integrate: You can drag with the right mouse button to create the whole integral from scratch or you can adjust one or two of the anchor knobs by dragging with the left mouse button.

An integral in MultiTrace consists of two anchor knobs, one left and one right of the peak. Each anchor has a time coordinate and an intensity coordinate. The intensity coordinate (along from bottom to top) can be anywhere below the profile but not above. Both anchors are connected with a straight base line and the area between the chromatographic profile and the base line is the integral area, which is filled in red. Negative areas are ignored, only positive areas are counted, i.e. those areas where the profile is above the integral base line.

Re-integrating from scratch: To re-create the integral from scratch, point the mouse on a reasonable place left of the peak, then press down the right mouse button, hold it down and drag the mouse to where you want the integral to stop anywhere right of the peak, then release the right mouse button. You may repeat this procedure as often as you need to be satisfied with the integral.

Note that MultiTrace on-the-fly repaints and re-integrates the new integral while you drag, including updating the integral values at the top of the graph.

Further note, that MultiTrace automatically fits the anchor points exactly on the profile whenever you point the mouse anywhere above the profile. This behaviour makes it easy to set integrals that are meant to have anchors on the profile. Do not try to exactly hit the profile with the mouse pointer, because this is just a waste of effort. Just point anywhere above the profile, drag the mouse and you are done.

If you intentionally want to create integrals with anchors below the profile, e.g. step-like integrals in case of co-eluting peaks, just move the mouse below the profile and MultiTrace will exactly follow your position.

Try re-integrating several times, both above and below the profile and get accustomed to the integration procedure.

Re-integrating using the anchors: You may left-click the small rectangular anchors and drag them to a new position. This feature is intended for small corrections. In most cases, re-integrating from scratch is faster, but moving the anchors is generally more accurate.

Please note that it is part of MultiTrace's concept that each ion of each parameter does have an integral. There is no way to delete an integral. You can just change an integral.

Synchronised integration of all ions

Having just trained the integration feature you will have noticed that whenever re-integrating the first ion, the second ion is re-integrated automatically, too. Both ion traces always automatically have integrals with the same time range, only the intensity axis is corrected to fit the profile. This drastically speeds up re-integration in case of MultiTrace picked the wrong peak or you want to cut off co-elutions or select only one half of split peaks. Generally, ions of the same mass spectrum of the same chromatography should have identical time ranges.

However, of course you are free to integrate the ions separately and independently. The easiest way to do so, is to first integrate the first ion, then integrate the second and third ion. You will see that only the first ion synchronises the other ions, but that the other ions do not synchronise the first one.

Should you want to turn off the synchronise feature, please click the chain icon in the upper right corner. Alternatively, you may hit the hot key "s" to toggle synchronisation on and off. However, usually, there should be no need to turn off the synchronisation. Just work from left to right instead.

Difference Integrals

In case of co-eluting peaks it is sometimes necessary to subtract parts of the integral area, i.e. in order to ignore small peaks which form shoulders on larger peaks.

If you hold down the Ctrl-key while re-integrating from scratch, you can create a difference integral. The subtract integral has to be inside the main integral. The colored integral area always shows the area being considered in the calculation, the subtract integral itself is white. You may re-integrate the subtract integral from scratch or by dragging its anchors.

Whenever you re-integrate the main integral from scratch, the subtract integral is deleted. If you want to modify the main integral, you have to use the anchors in order to keep the subtract integral.

Search time range

When selecting a previously untouched parameter, MultiTrace automatically looks for the largest peak inside the a given time window. This so-called search time range can be set and modified in the method definition file (but is not meant to be changed frequently during routine evaluations) and is indicated as bold gray line directly above the time axis.

The search time range has only influence on the automatic peak-picking. Naturally, you can manually select and integrate any peak, no matter whether inside or outside the given time ranges.

If you observe peaks wandering out of their given time window, then decide whether to correct the defined time window or to optimise the gas chromatographic conditions. In some laboratories, the method definitions are maintained by an administrator rather than modified by each operator. In that case, inform your administrator about the peak wandering out of its designated time window.

The search time range can be identical to the visible time range. In that case the gray line covers the whole time axis. It depends on the analytical conditions, whether a smaller search window and larger visible window is better than to search for peaks in the whole visible time window. It's your choice. Sometimes it is advantageous to see some surrounding, i.e. other parameters or isomers typically near to the current peak or matrix artefacts in order to decide about the identity of a peak.

Zooming

Shifting the time axis

When selecting a parameter, MultiTrace automatically displays the time range defined in the method definition file. However, of course the entire acquired time range is available for your perusal. Just click on the time axis, hold down the left mouse button and drag the time axis to shift the visible time window.

Stretch or compress

You can stretch or compress the time axis by dragging with the right mouse button. Click the right mouse button far right on the time axis, hold it down and drag it towards the center of the time axis and release the mouse button to compress the axis, so that a larger time range fits into the graph. Click a little bit right of the center of the time axis and drag to the right to stretch the time axis, so that a smaller time range fits into the graph and the time axis is zoomed.

Mouse zoom

You can simply drag anywhere in the graph with the left mouse button in order to zoom into that range. This is the standard Windows operation to select an area. Click the left mouse button and hold it down, drag anywhere towards the lower right and release the mouse button. MultiTrace will zoom the time axis to the selected area and auto-adjust the intensity axis so that all peaks will fit.

Thus, zooming with the mouse does only select the time range. The intensity range is always choosen by MultiTrace, so that all peaks fit into the graph. It does not matter how high or low you start or stop, only how far left or right you start and stop. See below for how to change the intensity axis.

If you accidentally started the mouse zoom operation and see the grayish-blue rectangle, you may cancel the operation by stopping at a position left and top of your start position.

Mouse cut

You can set a new intensity maximum for the intentity axis by Ctrl-drag with the left mouse button. Hold down the Ctrl-key, click the left mouse button at the desired intensity level, hold it down and drag the mouse a little bit to the lower right. MultiTrace indicates this special zoom operation by painting a grayish-blue rectangle over the whole time range. After releasing the mouse button, the new maximum of the intensity axis will be exactly the selected height that was indicated.

This method is perfect if you want to zoom in for a smaller peak. You can exactly control that the peak of interest will fit into the graph.

Zooming the intensity axis

You can stretch or compress the intensity axis by dragging the axis with the right mouse button. Right-click near the top of the intensity axis, hold the mouse button down and drag downward to compress the axis. Right-click anywhere in the lower half and drag upward to stretch the intensity axis. Dragging allows for smooth adjustments of the intensity axis.

Reset to default

You can reset the visible time window to the defined visible time range by double-clicking anywhere on the time axis. You can also reset both axes by simply selecting the same parameter again in the result table.


Continue with the next lesson of this tutorial: MultiTrace Tutorial: Evaluating a sample

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