Mass spectrometry (MS) is a useful tool for computer-assisted and automated analyses of the complex separations produced by GCxGC. In GCxGC-MS, each sample output of the GCxGC separation is analyzed by MS, e.g., time-of-flight (TOF) MS. Then, instead of a single value at each sample output, there is a multi-spectrum consisting of an array of (mass/z, intensity) pairs. These multi-spectra can be used in identifying unknown chemicals and in separating co-eluting peaks for more accurate quantification.
GC Image stores GCxGC-MS data in two files: a GCI file containing metadata (as described in File Input and Output) and a binary CDF (with .cdf extension) file containing the MS data. The CDF file conforms to the Analytical Data Interchange (ANDI) Protocol for Mass Spectrometric Data [ASTM E2077-00 and E2078-00]. The ANDI protocol is based on the network Common Data Format (NetCDF), developed at the Unidata Program Center and distributed by the University Corporation for Atmospheric Research. NetCDF is "a machine-independent format for representing scientific data." [www.unidata.ucar.edu] Most MS systems support export of data in ANDI/NetCDF format for non-proprietary data interchange. Using the GCI and CDF files, the GC Image file operations Open, Recent Images, Save, Save As, Close, and Exit work for GCxGC-MS images as they do for GCxGC images (described in File Input and Output).
GC Image supports importing GCxGC-MS data from ANDI files, Agilent MS and UV files, Shimadzu GCMS files, JEOL MassCenter files, and other formats (as listed in File Input and Output). To import GCxGC-MS data contained in an ANDI file (with .cdf extension) or in a Agilent MS file (with .ms extension), select the Import Image option from the File menu or click the Import Image button from the tool bar of the Image Viewer . GC Image prompts the user to specify the source file name (with .cdf or .ms extension) and the target file name. To maintain the original CDF file, the source and target file names should differ. GC Image also prompts for image metadata such as sampling rate, modulation cycle time, start time, and run time. If the image metadata values are imported from the source file, the value fields have values and are grayed. Grayed values may be overridden by double-clicking in the text box.
GC Image then imports the GCxGC-MS data and displays an image of the total intensity count (TIC) at each pixel, as pictured in Figure 1. The TIC is the sum of intensities of all mass/z values for the sample. Most of the image visualization, processing, and analysis tools for GCxGC data are available for GCxGC-MS data. Some of the tools specific to GCxGC-MS are described in this chapter.
|
| Figure 1: An image of the total intensity count (TIC) values for GCxGC-MS data. |
For GCxGC-MS data, the import dialog allows the user to optionally limit the maximum number of values in each multi-spectrum. GCxGC-MS analyses generate large data sets that may exceed the memory capacity of desktop systems, causing slow processing. One approach to processing GCxGC-MS data more quickly is to reduce the size of the GCxGC-MS data by retaining only the elements with the largest intensity values in each multi-spectrum and using sparse matrices to store the reduced multi-spectra.
For example, a GCxGC-MS image with 756x800 GCxGC samples and 215 intensity values in each multi-spectrum requires about one gigabyte of memory (using four byte integers for the mass/z and intensity values) as shown in Table 1. If only the 32 elements with the largest intensity are stored for each multi-spectrum, the memory requirement is reduced drastically to about 0.15 gigabytes as shown in Table 2.
| Mass/z values array | 756 x 800 x 215 x 4 = 520,128,000 Bytes | 496.03 MB |
| Intensity values array | 756 x 800 x 215 x 4 = 520,128,000 Bytes | 496.03 MB |
| Total memory required to store both the arrays: | 992.06 MB | |
| Mass/z values array | 756 x 800 x 32 x 4 = 77,414,400 Bytes | 73.82 MB |
| Intensity values array | 756 x 800 x 32 x 4 = 77,414,400 Bytes | 73.82 MB |
| Total memory for both the arrays | 147.64 MB | |
Of course, the gain in storage and processing efficiency must be weighed against the loss of data. Figure 2 shows the reduction of a multi-spectrum to retain only the values with the largest intensity in a sparse representation.
 |
| Figure 2: An example of multi-spectrum data reduction with sparse representation. |
GC Image imports mass/z values in the given number format, which may be integer or floating point. Some operations on multi-spectra (such as NIST MS Library Search) require integer mass/z values. The threshold for rounding up fractional values to the next largest integer can be set via Configure -> Multi-Channel. The threshold can be any non-negative value less than 1.0. The default threshold is 0.5, so that numbers with fractional values less than 0.5 are rounded to down and numbers with fractional values greater than or equal to 0.5 are rounded up.
The GC Image MS Viewer provides graphical and tabular views of a spectrum and other panes with additional information (e.g., as illustrated in Figure 3). If the tabular view or other panes are hidden, they can be shown by clicking the associated arrow pointing to the center of the window. Similarly, these panes can be hidden by clicking the associated arrow pointing away from the center of the window.
 |
| Figure 3: The MS Viewer GUI. |
In the graph (at the center of the window), the mass/z values are on the horizontal axis and the intensity values are on the vertical axis. The range for the horizontal axis is determined by the minimum and maximum mass/z values in all multi-spectra of the image (so, the same absolute scale is used for the horizontal axis in all the multi-spectra graphs). The domain for the vertical axis is from zero (or the minimum intensity if it is less than zero) to the largest intensity value in the selected multi-spectrum. The vertical axis is expressed as intensity value, percentage of total intensity, or relative percentage, depending on the setting of the radio buttons below the graph.
Mass/z labels are placed adjacent to selected large-intensity MS values. As the cursor is moved across the graph, the mouse-over tool-tip indicates the mass/z and intensity value, as in Figure 3. Click-and-drag with the right mouse button defines a zoom window for redisplay of a region of the graph. With the Reset button, the user can choose to reset (i.e., unzoom) the spectral axis, intensity axis, or both axes.
The tabular view (on the right side) has two columns: one for mass/z values and one for the corresponding intensity values in the multi-spectrum. The intensity values can be expressed as absolute values, as percentages of total intensity (so that all values sum to 100), or as relative percentage values (so that the largest valued entry is 100.00). The choice of value type is set by the radio buttons below the graph. The entries in the table can be sorted in forward or reverse order by clicking on the label at the head of the column to be used as the sort key. (Consecutive clicks reverse the sort order.) The total intensity count (TIC) for the multi-spectrum is shown below the table.
The Spectrum List, at the left side of the MS Viewer, contains one row per spectrum shown in MS Viewer and several columns:
The MS Viewer allows displaying and comparing multiple spectra in three layouts:
The MS Viewer allows the active spectrum to be copied in one of several formats using the Copy to Clipboard button on the tool bar. The spectrum can be copied as a table or string (full range or selected range) and the MS graph can be copied as an image.
GC Image supports selecting multi-spectra for viewing in three modes:
As illustrated in Figure 4, the Image Viewer palette contains a button to set the cursor mode with a pull-down MS mode selector.
 |
| Figure 4: MS mode selection. |
In Point MS mode, when the user clicks the left mouse-button on a point in the Image Viewer (e.g., as in Figure 1), GC Image retrieves the multi-spectrum for the corresponding sample and displays it in a popup window, as in Figure 3 .
In Peak MS mode, when the user clicks in a blob in the Image Viewer, GC Image displays the multi-spectrum of the datapoint within the blob that has the largest total intensity count (TIC), i.e. , the multi-spectrum at the blob apex. This mode is accessible only after blob detection has been performed.
In Blob MS mode, when the user clicks in a blob in the Image Viewer, GC Image displays the sum of the multi-spectra of all the datapoints in the selected blob. In computing the sum, the mass/z values are rounded to the nearest integer. This mode is accessible only after blob detection has been performed.
Using the Multi-Channel dialog via Configure -> Configure Settings, the user can specify whether successive selections of multi-spectra are presented in multiple windows (in which case a new window is opened for each successive multi-spectrum) or in a single window (in which case the previous multi-spectrum graph is replaced in the same window by the new multi-spectrum). The Windows pull-down menu in the Image Viewer allows switching focus between multiple windows that may be open.
The MS Viewer allows the user to subset the multi-spectrum, by selecting one or more mass/z subranges. Multi-spectrum subranges are particularly useful for generating a selected ion chromatogram (SIC), described below. To specify a subrange of mass/z values (including a subrange with a single mass/z value), the user clicks-and-drags the left mouse-button in the graph area of the MS Viewer. Additional subranges can be added by depressing the Control-Key during click-and-drag. Subranges also can be added by entering the minimum and maximum mass/z values and then clicking the Add button in the Add Range dialog of the MS Viewer. Overlapping subranges are merged. The reset button or left mouse-button click without the control key clears any previously selected subranges. Figure 5 illustrates a multi-spectrum with two subranges specified.
 |
| Figure 5: A multi-spectrum in the MS Viewer with two subranges selected. |
The MS Viewer has a button to generate a selected ion chromatogram (SIC). A SIC is constructed with each pixel having the value of the summed intensities in the indicated mass/z subrange(s) of the multi-spectrum at the corresponding datapoint. An example SIC for the mass/z subrange 120-134 is illustrated in Figure 6.
To generate a SIC, first select a spectrum (for a pixel, peak, or blob) for display in the MS Viewer window. Then, in the MS Viewer, select one or more mass/z subranges. (A subrange may have a single mass/z value.) Finally, click the Open Ion Image button in the MS Viewer control area.
Using the Multi-Channel dialog via Configure -> Configure Settings, the user can specify whether successive ion chromatographic images are presented in multiple windows (in which case a new window is opened for each successive ion chromatogram) or in a single window (in which case the previous ion chromatogram is replaced in the same window by the new ion chromatogram image). The Windows pull-down menu in the Image Viewer allows switching focus between multiple windows that may be open.
 |
| Figure 6: An example ion chromatogram image for the mass/z subrange 90.5-91.5. |
The user can save a SIC image to a separate file through File -> Save As, thereby invoking a file browser to specify the location and name of the GCI (.gci) file. An accompanying ANDI NetCDF (.cdf) file also is saved.
GC Image supports addition and subtraction of spectra, as well as addition, subtraction, multiplication, and division of a scalar value. These operations can be used for spectral background removal, averaging spectra, etc.
To add two spectra:
The resulting spectrum is set to active and displayed in the MS Viewer.
To subtract two spectra:
The resulting spectrum is set to active and displayed in the MS Viewer.
Scalar operations on a spectrum are performed on a mass/z basis. The supported operations are add, subtract, multiply, and divide.
To perform a scalar operation on a spectrum:
The resulting spectrum is set to active and displayed in the MS Viewer.
The MS Viewer has a button to initiate a library search for the selected multi-spectrum. The Lookup Library button invokes a library search to match the selected multi-spectrum. A library search also can be performed using multi-spectrum subranges.
GC Image interfaces with the National Institute of Standards and Technology (NIST) MS Search Program and through it can search the NIST/EPA/NIH Mass Spectral Library, the NIST/EPA/NIH Mass Spectral Demonstration Library, or any multi-spectral library that is format-compliant. The CDROM installs the MS Search Program (Version 2.0) and the NIST/EPA/NIH Mass Spectral Demonstration Library, which contains 2400 compounds, a small fraction of the full NIST/EPA/NIH Mass Spectral Library. For information about purchasing the full NIST/EPA/NIH Mass Spectral Library, click here .
GC Image supports both identity search and similarity search of the designated library (or libraries). If the unknown compound is likely to be in the library, identity search is the quickest way to find it. Identity search uses a very efficient method of selecting a small set of spectra for subsequent spectrum-by-spectrum comparison. The two types of identity search are Quick Search and Normal Search.
Similarity search may be used for spectra that probably are not in the library. The four types of similarity search are: Simple, Hybrid, Neutral Loss, and MS/MS.
The NIST MS Search Program returns a "hit list" of matched chemical compounds from the library and several factors for each matched compound. GC Image displays these in a table, as illustrated in Figure 7. Compounds in the hit list are listed with the following attributes:
 |
| Figure 7: Example MS Search Hit List Table. |
By default, the hit list contains ten compounds with the highest match factor. The user can configure the size of the hit list using the Library Lookup panel in Configure Settings, illustrated in Figure 8. The user also can designate the libraries to be searched and the type of search.
 |
| Figure 8: Library search configuration pane. |
GC Image can use the library search to automatically set the compound names for blobs. Clicking the Search MS Library button on the Image Viewer tool bar or selecting Filter -> Search MS Library from the menu submits one or more blob (peak or integrated) multi-spectra for library search and sets the compound name of each submitted blob to that of the library compound with the largest match factor for that blob's multi-spectrum. After library search for a blob, GC Image's maintains the "hit list" for that blob, which can be accessed via the blob metadata popup, as pictured in Figure 9.a. The user can choose interactively any compound in the hit list as the name of the blob, overriding the initial identification of the compound with the largest match factor, as illustrated in Figure 9.b.
 |
| Figure 9.a: Blob Metadata popup with a button to access the hit list. |
 |
| Figure 9.b: An example hit list for a blob. |
The library search be made on all blobs or on all selected blobs and the search can be made using either the peak multi-spectrum or the integrated multi-spectrum of each blob (configured by the user, as illustrated in Figure 8 ). The user also can set the configuration so that library search is performed automatically after blob detection.
GC Image has several utilities for customizing chemical identification based on multi-spectral information. In particular, CLIC expressions can combine MS library search and other MS-related functions to create custom identification rules that can be combined with retention-time templates and graphical objects. CLIC expressions and the CLIC tool are described in Chemical Identification. The tools to specify and use characteristic ions, including both qualifier and quantifier ions are important features for customized spectral analysis.
Qualifier and Quantifier Ions can be used to perform precise qualification or quantification of chemicals using Qualifier Ion ratio tests and Quantifier Ion response calibrations. The Qualifier/Quantifier Ion Table is available in the blob metadata dialog as well as the template peak metadata dialog. Records in the table consist of four components: Qualifier Ion Range, Quantifier Ion Range, Expected Ratio (intensity in the Qualifier Ion Range divided by the intensity in the Quantifier Ion Range), and % Tolerance (allowed with respect to the Expected Ratio).
To edit Qualifier/Quantifier Ion Table, select a blob and open its properties dialog (shown in Figure 10). Type in the desired values for new Qualifier/Quantifier record(s), using the Multi-Spectrum buttons for the Qualifier and Quantifier ions to indicate the desired ions in the multi-spectrum for that blob. Enter values for the Expected Ratio and % Tolerance. Click OK to save the blob information including the Qualifier/Quantifier Table and any other blob metadata. As with other blob metadata, Qualifier and Quantifier records are copied with blobs into template peaks and from template peaks into matched blobs.
 |
| Figure 10: Blob Properties Dialog with Qualifier/Quantifier. |
Elements of the first four rows of the Qualifier/Quantifier Ion Table and computations made with elements of the first four rows of the Qualifier/Quantifier Ion Table are available in the blob table.
To view the Qualifier/Quantifier Ion in the Blob Table:
 |
| Figure 11: Blob Table with Qualifier/Quantifier. |
Note that the Quantifier ion can be set without a Qualifier or Expected Ratio, in which case the Quantifier Response is available, but no ratio is computed and no test is performed.
Qualifier tests are used in template matching to screen potential matches. Each test determines whether or not the actual ratio of the Qualifier to Quantifier responses for potentially matching blob are greater than (Expected Ratio – % Tolerance * Expected Ratio) and less than (Expected Ratio + % Tolerance * Expected Ratio) from the template peak. If any Qualifier test in the template peak's Qualifier Table fails, then the blob does not match.
All Qualifier and Quantifier values can be used in CLIC expressions, including Qualifier, Quantifier, Expected Ratio, Tolerance, Qualifier Response, Quantifier Response, Qualifier Ratio, and Qualifier Test. This includes all Qualifier/Quantifier records, not just the first four records listed in Qualifier/Quantifier Ion Blob Features. Values in Qualifier and Quantifier records are indexed with square brackets, e.g., Qualifier[5] refers to the Qualifier ion for the fifth record..
The Analysis section in the Blob Metadata dialog supports an Analysis CLIC expression, labeled aCLIC, and a Qualifier CLIC, labeled qCLIC. The result of evaluating the aCLIC expression can be seen in the aCLIC Result feature in the blob table. The result of evaluating the qCLIC expression is used to constrain template matching, i.e., if the qCLIC expression evaluates to false with respect to a potentially matching blob, the blob is not matched.
GCxGC-MS images have an intensity value for each spectral channel at each data point. Spectral colorization maps various spectral channels to different color groups, revealing spectral information embedded within large and complex GCxGC-MS data.
A spectral color map can be applied to an image shown in 3D View. With spectral colorization, each point on the 3D surface is colorized according to the spectrum at that datapoint. The color map acts as a filter of the spectrum at each point. Each spectrum is composed of a series of spectral channels with varying intensities. At each channel, if the intensity of a spectrum is greater than zero and the channel is within an assigned spectral range of the color map, the pixel on the 3D surface is shown with the map color for that range. If more than one color is to be shown for a spectrum, the colors are mixed proportionally according to their corresponding relative intensities. For example, a spectral color map shown in the Figure 12(a) when applied to the mass spectrum shown in Figure 12(b) produces the color yellow because the intensities in the spectral range colored red and the intensities in the spectral range colored green are equal and an equal mixture of red and green is seen as yellow.
 |
| Figure 12: An example spectral color map |
The 3D View tool allows user control over the spectral color mapping. In the 3D View control panel, the user can choose to colorize the surface based on only TIC values or spectra. Clicking on the Spectra option under Color Map opens the Spectral Color Map configuration dialog, as shown in Figure 13.
 |
| Figure 13: The Spectral Color Map configuration dialog |
The Spectral Color Map configure dialog supports:
After finishing the configuration, click OK to apply the color map. A message is displayed prompting to either apply the color map or do it later. Choosing Yes applies the color map. Choosing No saves the settings without applying the color map. Then the color map can be applied later by choosing Apply Above Settings in 3D View Controls. Figure 14 shows the 3D View of a diesel sample colorized with a spectral color map based on ASTM Standard D2786-91.
 |
| Figure 14: A diesel image colorized with a spectral color map (red - alkenes, cyan - 1-ring, green - 2-ring, magenta - 3-ring, yellow - 4-ring, blue - monoaromatic). |
The GC Image MS Cube tool supports exploring GCxGC-MS data in a 3D space whose dimensions are Column I, Column II, and MS. The MS Cube allows the user to quickly browse through SICs of any orthogonal slice (Col IxCol II, Col IxMS, Col IIxMS) and the spectrum at any point to discover interesting patterns. GCxGC-MS data is displayed as a cloud of points in the 3D space. Each point represents an intensity of a nominal mass in a spectrum. Because of the limits of memory and graphics card, the software filters out the low intensities and displays at most 5 million data points. The suggested minimum hardware configuration for MS Cube is a 256MB OpenGL-capable graphics card and a color monitor with 1280x1024 (or higher) resolution.
The MS Cube tool is available through View -> MS Cube from the GC Image menu. Figure 15 shows a diesel image displayed in the MS Cube. The interface consists of four panes: Cube View for displaying spectral data points in 3D, 2D View for displaying the current orthogonal slice, Spectrum View for displaying a spectrum of interest, and a control pane.
 |
| Figure 15: The MS Cube. |
The software supports rotation and zoom on the Cube View in the same manner as in the 3D View tool, through the mouse actions or the Viewing Angle control. The number of data points visible on the Cube View is controlled by adjusting the threshold slider in the Visibility control. There are two ways to set the visibility threshold:
The software displays an orthogonal slice (Col IxCol II, Col IxMS, Col IIxMS) of the MS Cube in the 2D View panel. The Cube View shows a red Slice Plane that indicates the location of the slice in the 3D space. The orientation of the Slice Plane can be controlled by choosing the corresponding radio button options in the Slicer control:
The current slice position can be adjusted by dragging the slider in the Slicer control. Clicking the animation button at the right of the slider starts the Slice Plane in progressive motion. Clicking on the button again stops the animation. The software also supports dragging the slicer in the Cube View. Select Enable 3D Slider in the Advanced control panel to turn on this feature. Once this feature is enabled, a red-ball cursor is displayed on the axis along with the slice plane, as shown in Figure 15. The user can drag the cursor along the axis to move the slice plane. Clicking on the axis moves the slicer to the corresponding location. Zoom in Cube View can improve the mouse-drag precision.
By default, the 2D View only displays the projection of visible data points to the current slice plane. The software also supports displaying the current SIC image on the slice plane in Cube View and/or the 2D View in real time. This feature is turned off by default because intensive computation is required. Options in the Advanced control panel are available for enabling the SIC image display on the slice plane and the 2D View individually:
If the current slice is a Col I x Col II slice other than the TIC (i.e., a SIC), then the slice image can be opened in the Image Viewer as an independent SIC window by clicking on the pop-up button at the top-right of the 2D View.
Clicking in the 2D View displays the point spectrum at the corresponding location. The Spectrum View is located at the bottom-left of the interface. Clicking on the pop-up button at the top-right of the Spectrum View opens the spectrum in a separate MS View with more advanced features.
The software supports the display of a set of static Col I x Col II SICs in the Cube View. In addition, a static TIC plane is always placed at the zero on the Z axis, as shown in Figure 16. The user can manage these static SICs in the SIC Table in the control pane. The SIC Table shows the MS location of all the static SIC images. The user can add or delete static SICs from the SIC Table. To add a static SIC, simply move the Slice Plane in the Col I x Col II dimension to the desired spectral slice and click Add in the SIC Table. Then, a static SIC plane is added to the Cube View and the SIC Table. To remove a static SIC, select the desired SIC in the SIC Table and click Remove. Then, the static SIC is removed from Cube View and the SIC Table. With Enable 3D Slider, right clicking anywhere on the axes or the red-ball cursor brings up a menu for Add/Remove SIC. The list of SICs in the current SIC Table can be saved to a CSV file and reloaded later.
GC Image supports extracting multiple reaction monitoring (MRM) data with a user-defined precursor ion list using Importer or importing Agilent Mass Hunter data files acquired with a single scan group of one of the MS/MS level types — MRM, Precursor Ion, Product Ion, Neutral Loss, and Neutral Gain — through MassHunter Data Access Component (MHDAC) 2.0. The following restrictions exist due to the limitations of the MassHunter DAC:
The software requires MS level data to perform other operations. If MS level data is not available in a MassHunter data file, the software reconstructs the MS level data based on the scan type as follows:
The software stores GCxGC-MS/MS data in three files: a GCI file containing metadata, a binary CDF (with ".cdf" extension) file containing the MS data, and a binary CDF (with "_ms2.cdf" extension) file containing the MS/MS data. Users can view MS/MS spectra in the same manner as viewing MS spectra. The software displays all corresponding MS/MS spectra when displaying a MS spectrum (point, peak, or blob), in the same MS Viewer. Each spectrum is displayed with a unique color, as shown in Figure 16.
 |
| Figure 16: MS Viewer with MS and MS/MS spectra. |
A MS/MS SIC can be generated using the MS Viewer similarly to how a MS SIC is generated. To generate a MS/MS SIC, first view a spectrum in MS Viewer. Then, change the active spectrum to one of the MS/MS level spectra and select a range. Finally, click the Open SIC button on the toolbar to open the SIC. The software can export a SIC from MS/MS to a CSV file in the same manner as from MS. The MS/MS SIC also can be saved as an image file. When a MS/MS SIC is saved, only the spectra for the selected MS/MS ion is saved. All MS and any other MS/MS scan information is discarded.
GC Image™ Users' Guide © 2001–2010 by GC Image, LLC, and the University of Nebraska.