13 Quantitation Wizard - University of Colorado Denver · 2010-07-29 · Quantitation Wizard 1 ... the “internal standard” name and its corresponding MRM transition of ... enter

Quantitation Wizard 1

Quantitation Wizard This exercise is designed to familiarize you with the Quantitate section of Analyst® Software. This tutorial utilizes sample data provided in the Example project folder. However, you may use data collected during earlier lab exercises.

Summary of Steps

In this tutorial you will learn the following:

1. Building a Results Table using Quantitation Wizard

2. Viewing and Processing the Results Table

Editing Columns Audit Trail Prompt Calibration Line Peak Integration & Integration Method Statistics Build/Execute a Query Build/Execute a Sort Build/Execute a Metric Plot Formula Column

3. Analyst Classic and IntelliQuan Integration Algorithms


13.1 Building a Results Table Using the Quantitation Wizard

Change to the Example project in Analyst® Software. Double-click on Quantitation Wizard in the Analyst® Software navigation bar.

The Select Samples dialog, as in Figure 1, appears.

Figure 1 - Selecting batches of samples for quantitation within Analyst Software.

Select the batch of samples to process from the available batch files (QuantData.wiff in the Triple Quad folder or other data). Choose the files to analyze from the list of data files in the batch and add them to the selected sample list (if using QuantData.wiff, select Add All). Click Next at the bottom of the window.


The Select Settings & Query dialog appears. In this dialog set the Settings to Use to “Default” and the Default Query option to “None” as shown in Figure 2 (these will be defined later in the tutorial). Click Next at the bottom of the window to move to the next phase in building the method.

Figure 2 - Select Settings & Query dialog for creating a quantitation set.

The Select Method dialog is now open. Three different options are available: Choose Existing Method (to select an existing method from the current project), Create New Method (enter a name in the box) and Create Automatic Method (to set the integration parameters from a chosen data file, then automatically integrate chromatograms to generate peak area data. This option is only available for the Analyst Classic and IntelliQuan MQII algorithms. These algorithms will be defined later in the tutorial).

Select Create New Method and enter the name “Training” (Figure 3). Click Next.

Figure 3 – The Select Method dialog for creating a quantitation set.


The Select Representative Sample dialog appears. In this dialog, highlight or scroll to a file that best represents all compounds to be quantitated, typically a middle concentration standard or API3-016 file in QuanData.Wiff (Figure 4). Note that the Sample Type, Analyte Conc. and Internal Std. Conc. columns contain information. These columns will indicate “Unknown” and be empty if the information was not entered in the Quick Quant option of Acquisition Batch prior to running the sample set (see Agilent capLC Tutorial -- Running the Acquisition Method). The concentrations will have to be entered in the final results table after the peaks have been integrated.

Click Next.

Figure 4 – The Select Representative Sample dialog with data file-appropriate data selected.

The Define Peaks dialog appears. In the Internal Standards and Analytes section define the name in the Name box, and the corresponding MRM or SIM transition in the Q1/Q3 box (Figure 5). The Sum Multiple Ions box, if checked, indicates that more than one ion or mass range is to be summed upon integration.

In this example, the “internal standard” name and its corresponding MRM transition of 420.0/220.0 and “analyte” with an MRM transition of 400.0/200.0 are selected from the drop down menu (Figure 5). The names in the drop down menu was entered in the Quick Quant option of Acquisition Batch prior to running the sample (see Agilent capLC Tutorial -- Running the Acquisition Method).


Figure 5 - The MRM transition of the internal standard defined in period 1.

In the column heading ‘Internal Standard”, assign an internal standard to the analyte(s) if present. In this example, “internal std.” was selected from the drop down menu.

Note: The names will not appear in the drop down menu if Quick Quant was not used before data acquisition. If there are no names to choose from, enter a name for each compound and select the corresponding Q1/Q3 from the drop down menu in this table.

Set the Default Number of Smooths. The valid range is 0 to 10 (this can also be changed after the peaks have been integrated).

If additional MS experiments and/or periods are present, repeat steps 5 and 6 for each. Click Next when you are finished.


The Define Integration dialog for the first peak, the internal standard (in this example), appears (Figure 6). Here the baseline noise is already defined and the peak has been automatically integrated. Set the RT Window (retention window) for the internal std. peak to 30.0 sec.

Figure 6 - The Define Integration dialog for the internal standard.

If the desired peak was not automatically found, select the background range (Figure 7) and the peak of interest (Figure 8) by making a selection in the chromatogram and clicking the corresponding icon in the top left hand corner of this window.

Figure 7 - Example of a background range selection.


Figure 8 - Example of a peak selection.

Click the Advanced button to open a dialog with more advanced integration parameters (Figure 9). The displayed Advanced dialog box will depend on the integration algorithm (see later section) selected prior to starting the Quantitation Wizard .If applicable, enter a Bunching Factor of 2 and the Number of Smooths as 2. Make sure the Concentration units match that of the standard, in this example ng/ml. Click OK to review the new peak integration.

Figure 9 - Advanced integration parameters for IntelliQuan (left) and Analyst Classic (right) .


Review the new peak integration by magnifying the baseline using the vertical (Figure 10). If the new integration parameters are suitable, click Next to move on to integrating the next peak.

Figure 10 - Integration window for the internal standard with the baseline magnified.

Repeat steps 9-12 for each peak to be used in the quantitation method. In this tutorial the only other peak to be used is that of the analyte. Make sure that the Concentration units match that of the standard, ng/ml. After defining the integration parameters for the analyte, click Next to go to the final phase of building the quantitation method.

In the Specify Calibration dialog, define the Fit to be “Linear”, the Weighting to be “1/x” and the Regression parameters to be based on Area (Figure 11).

Figure 11 - The Specify Calibration dialog.

Click Finish to complete the quantitation method. The sample files added above are automatically integrated and a table of results is generated. Save the new results table as Training. Proceed to next section to navigate through the results table.


13.2 Viewing and Processing the Results Table In this section, you will learn about:

1. Viewing and editing columns of the table and the Audit trail prompt.

2. Viewing a calibration line.

3. Viewing peak integration and updating integration methods.

4. Viewing statistics.

5. Building and executing a query.

6. Building and executing a sort

7. Building and executing a Metric plot

8. Using a formula column.

Viewing and editing the columns of the results table

If the results table is not open, double-click on Review Results Table in Analyst® Software

In the resulting dialog box, select the previously saved Training.rdb file. The results table appears (Figure 12).


Figure 12 - The unedited Training results table and the right click menu

From the right-click menu, select Table Settings and then Edit (Figure 12).

The Table Settings dialog reappears. Click Columns in the dialog, then Edit and the Results Table Columns dialog appears (Figure 13). In this dialog, you can define the columns that will appear in the results table. The columns are separated into 6 classes (Sample, Analyte, Internal Standard, Record, Formula and Custom) in the drop down menu at the top of this window. If a custom column was added in Acquisition Batch prior to running the sample, the custom column can be selected from the list.

In this menu, choose Sample and in the resulting dialog, uncheck Sample ID (this removes this column from those visible in the results table).


Figure 13 – The Results Table Columns dialog for a results table.

Choose Analyte in the menu. In the resulting dialog, uncheck Analyte Peak Height. Adjust the Precision of the Analyte Peak Area & Analyte Concentration from 3 to (Figure 14). These columns will now display their information to 5 significant figures.

Figure 14. Columns available for the Analyte in a results table.

Choose Internal Standard in the drop down menu and repeat step 4. Click OK to use the new settings and then Done to return to the results table with the adjusted columns (Figure 15).


Figure 15. Results Table with adjusted columns

To save these new column table settings, right click and choose Table Settings then Export to New Table Settings (Figure 15). Enter a name for this table setting. This setting can now be accessed whenever a new results table is created (in this project only) by using the same right click menu and selecting the desired table name under the gray bar line or by selecting it in the ‘Settings to Use’ drop down menu of the Select Settings and Query dialog (Figure 2).


The Audit Trail

Click Audit Trail in the Table Settings dialog and then click Edit (Figure 16).

Figure 16 - Table Settings dialog for a results table.

In the Audit Trail Settings dialog (Figure 17), uncheck the “Enable audit tracking” box and all options are grayed out. Click OK and Done to accept the change and disable the audit trail

Figure 17 – The Audit Trail Settings dialog for a results table.


To enable audit trail, check the “Enable audit tracking” box. (Figure 18). You can add or remove reasons from the audit trail list (using New Reason or Remove, respectively) as well as have the ability to allow or not to allow users to specify custom reasons. For this tutorial, uncheck the box next to Prompt user for reason. This removes the audit trail prompt that appears every time the results table is adjusted, though any changes to the results table are still logged in the audit trail but without a reason and signature defined. Click OK and Done to use the new settings.

Figure 18 – The Audit Trail Settings dialog for a results table.

To view the audit trail, click on Tools – Audit Trail – Show or click on the icon


Viewing a Calibration Line

Prior to viewing the calibration line, the files to be used as standards must be specified in the Sample Type column using the drop down menu. The corresponding concentration for each standard must be entered in the Analyte Concentration column. In this tutorial example, these columns are automatically filled because this information was included in the Quick Quant option of Batch Acquisition (see Agilent capLC Tutorial -- Running the Acquisition Method for more info). prior to running the samples.

To view the calibration line, click on Tools – Calibration – select Window or Pane. You could

also click on the icons to display the calibration line as a pane or window, respectively.

Window opens a new window for the calibration and Pane opens a new pane for the calibration at the bottom of the results table.

Click the Calibration – Pane icon.


Figure 19 - Calibration pane of the Quantitation section of Analyst Software.

In the calibration pane, you can view individual analyte calibration curves (use the drop down Analyte menu) as well as change the regression used on the data (Figure 19). In this example, there is only one analyte in the Analyte drop down menu.

Change the regression used for the data by clicking the Regression button in the window (Figure 19). In the Regression Options dialog, change the Fit to Quadratic and the Weighting to None. Click OK to view the changes.

The Regression Options dialog.

The calibration line now uses the new regression settings, which causes the equation for the calibration line and ‘r’ value to change. To use these new settings, click the Accept button. For this tutorial select Revert to use the previous settings, then close the calibration window.


Viewing Peak Integration and Updating Integration Methods The integrated peaks can be viewed in a format much similar to the calibration line, that is, as a window or pane. Both window and pane can be accessed by Tools – Peak Review or by

clicking on the icons .

To view the integrated peaks, click on the desired file then click on the Peak Review Window icon. A new window opens up with the selected file displayed as the active file (bold font). To customize the display, right click and choose Options (Figure 20).

Figure 20 - The Peak Review Window.

The Peak Review Options dialog appears (Figure 21). In the Appearance section of this dialog set Num. Rows to 1 and Num. Columns to 2. In the Automatic Zooming section, choose Zoom Y axis to 100 % of largest. Click OK to use the new settings.


Figure 21 - Peak Review Options dialog in Quantitate.

These settings apply to this Peak Review window only and will revert to the default settings when a new Peak Review window is opened. To change the default settings, click on Tools – Settings – Quantitation Peak Review Settings and make the changes in this box.

Toggle the Show or Hide Parameters icon (Figure 22) at the top of the Peak Review window to view or hide the peak integration parameters. Depending on the integration algorithm chosen (Analyst Classic or IntelliQuan, see later section) prior to going through the Quantitative Wizard, the displayed integration parameters will vary.

Quantitation Wizard

Figure 22 – The Peak Review pane with the new option settings and peaparameters.

If applicable, change the Bunching Factor to 2 and the Num. Smooths to 1 inparameters and click Apply to re-integrate the peak. Click Accept to save thparameters for this sample. When the integration of a data file is altered, thecolumn in the Results Table is checked for that sample. However, the other use the original integration method.

To apply the new integration parameters to all the data files in the results tathe Peak Review pane and select Update Method. All the data files will be inew method. Note that this change applies to the selected MRM only

A method can also be updated while viewing the results table by selecting thicon in the Quantitate tool bar. When you select this icon, you can change thparameters for each compound, the fit for the calibration line and the compofor the data files, i.e. the peak names and corresponding MRM or SIM transcompound.

To use the Manual Integration Mode, click the icon in the Peak Reviewand drag the mouse from the base of one side of the peak of interest to the onow manually integrated and the integration parameters used previously areinactive (Figure 23).

Peak Integration Parameters

19

k integration

the integration e new integration Record Modified data files will still

ble, right-click in ntegrated using the

e Modify Method e integration nent i\nformation ition for each

Window. Click ther. The peak is greyed out and


Figure 23 - Manual Integration.

Integrated peaks can also be viewed by using the Slide Show Peak Review. Right-click on the Peak Review pane or window, select Slide Show Peak Review to display the Peak Review Slide Show dialog

Click the Review peaks button: . The Peak Review window automatically scrolls through the data files with the specified delay.

To stop reviewing the peaks, click the Cancel icon: . You can also pause the slide show

with , go back to the beginning of the results table with or view a previous Peak

Review page with . Alternatively, the Peak Review for the first or last data file can also be viewed by selecting this option in the right mouse button menu while in the Peak Review pane or window.

Print the chromatograms for all the data files by selecting Print Pane from the File menu or, alternatively, print just the chromatograms that are being viewed by selecting the Print Window option.


Viewing Statistics

You can view statistics for Standards and QC’s by clicking the statistics icon in the Quantitate tool bar.

Click the Statistics icon in the Quantitate tool bar while viewing the results table. The Statistics window (Figure 24) appears.

Figure 24 - The statistics window of Analyst Software.

Set the Statistics Metric to Concentration. In the statistics window each analyte is viewed separately by selecting the appropriate Analyte Name.The standards and QC samples are also viewed seperately by selecting the appropriate Sample Type


Building and Executing a Query

A query is a method of selecting only those records that meet certain criteria. You create a query using textual or mathematical selection criteria. There are two methods you can use to create a query. You can use the Select Settings & Query dialog in the Quantitation Wizard (Figure 25) or you can create a query from the Results Table (Figure27).

Figure 25 - Select Settings & Query dialog box in Quantitation Wizard

If you select the option Create New Standard Query in the Quantitation Wizard, the Create Query (Figure 26) dialog box appears. Specify either concentration or name for QC’s and Standards and the Max. Variation % in the columns. The concentration column will be automatically filled if the Quick Quant option was used in Batch Acquisition. Click Next. The query results will be shown as a ‘Pass or Fail’ entry in the Standard Query Status column of the Results Table.

Figure 26 -Create Default Query dialog box

If you select the option Select Existing Query, it is executed in one of two ways. If the ‘Execute Query as a Standard Query’ box is checked, the query results will be shown as a ‘Pass or Fail’ entry in the Standard Query Status column of the Results Table. Otherwise, the


Results Table will show only the rows matching the criteria specified in the query. If the query is so selective that no record meets your criteria, the table displays no data.

If you create a query from the Results Table (“Non-Standard Query”), only the rows meeting the criteria you specified are displayed. Example of a “Non-Standard Query”: Right-click and select Query, then New. The Query Edit dialog appears (Figure 27).

Figure 27 – The Query Edit dialog for the result table.

In the Field Group option box choose “Record” and in the Field box alongside choose “Accuracy”. In the box below Field Group choose the option”is less than” and in the box alongside type in 90. Click the Add option on the right side of the dialog to save this part of the query.

Repeat step 2 above except choose the option “is greater than” and in the box alongside type in 105, then click Add to add this part of the query to the list.

Change the And statement in the query to Or by highlighting the middle line And in the query and choosing the Or option on the right hand side of the dialog. Type Acurracy Test into the Name box and then click Save/Execute to both save and execute the query on the results table.

The query is saved, the results table is interrogated, and the data files that have an accuracy either below 90 or above 105 remain in the results table (Figure 28). To review all the samples of the original results table, click the Show All Samples icon above the results table.


Figure 28 - Result of the Accuracy Test query on the Training results table.


Building and Executing a Sort

The Sort dialog allows you to rearrange the data files in the results table in a specified way. You can sort the data on three criteria at once.

Right-click and select Sort, then New to open the Sort dialog (Figure 29).

Figure 29 – The Sort dialog for a results table. For the first sort, choose Sample for the first box entitled Group, followed by Sample type in the Column box below. Select Descending for the sort direction. Repeat step 2 for the second sort except select Analyte in the Group box, Analyte concentration in the Column box choose and Ascending for the sort direction.

Type in Sample type, concentration in the Name field (Figure 30 ) and click Save/Execute to both save and run the sort at the same time.

Figure 30 - Sample Type versus Analyte Concentration sort.


The data files in the Results Table are then rearranged according to the sort parameters (Figure 31).

Figure 31 - Rearranged Results Table after sorting by Sample Type and Analyte Concentration.


Building and Executing a Metric Plot

The Metric Plot produces an X-Y graphic plot from two selected columns in the results table. Upon executing a metric plot, a new window appears with one column plotted against the other. Typically this type of plot can be used to graphically show Concentration vs. Time (if available) plots, Retention Time/Internal Standard stability or how the chromatography is changing during the analysis of a batch of samples (as shown in the column test example below).

Right-click and select Metric Plot then New to open the Metric Plot dialog (Figure 32).

Figure 32 - Metric plot dialog of the Result Table

In the Metric Plot dialog, in the box entitled X Axis select “Internal Standard” for the Group followed by IS Peak Area in the Column box below. In the box entitled Y Axis, select Internal Standard for the Group followed by IS Peak Height in the Column box below.

Type in Column test in the Name box and Linear in the Regression drop down menu of Show (Figure 33) then click Save/Execute to both save and run the Metric Plot at the same time. A metric plot pane is appears, with Internal Standard Peak Height plotted versus Peak Area.


With the mouse button on the metric plot pane, select the Point Legend option. A table displaying the point legends appears on the left hand side of the pane (Figure 34).

Figure 33 - Column test metric plot.

Figure 34 - Results table window showing the Column Test metric plot pane.


Using a Formula Column (Optional Section)

You can use a formula column to process data in the columns of a results table using a mathematical formula similar to the Microsoft Excel program or other spreadsheet programs. The example below shows a way to test the chromatography during the analysis of a batch of samples.

Right-click in the top left hand corner of the results table and select Add Formula column. A new Formula #1 column appears at the end of the results table.

Click on the new formula column with the left mouse button and type “=IS Peak Area/IS Peak Height” in the Formula field (in the top left hand corner of the results table), then hit Enter to save this formula and perform the calculation. You can also ‘click on’ instead of ‘typing in’ the column headings when setting up the equation for the formula. In this example, the data in the column IS Peak Area is automatically divided by the data in the column IS Peak Height and the results are shown in the new formula column (Figure 35).

Figure 35 - Results table showing a new formula column.

To change the name of the formula column, double-click on the column to display the New Title dialog (Figure 36). You can rename the column heading in this dialog. To delete the formula column, click the Delete Formula Column icon at the top left side of the results table.

Figure 36 - New Title dialog for renaming a formula column.

To save all the changes to the results table format, right-click in the results table, then select Export to New Table Settings. Type in a name in the New title dialog. The table settings will now be saved and available to use when building a new quantitation method within the same project.


13.3 Analyst Classic and IntelliQuan Algorithms There are two Integration Algorithms available to Analyst® Software users. The Analyst Classic in version 1.4 has all the same features familiar to Analyst® Software users whereas the IntelliQuan algorithm incorporates some features from the MacQuan software. The algorithm must be specified prior to using the Qunatitation Wizard.

To specify the algorithm to be used for integration, click on Tools-Settings – Quantitation Integration Algorithm and choose between Analyst Classic or IntelliQuan. The Quantitative Wizard will automatically use the chosen algorithm selected.

Figure 37. – Choosing the quantitation algorithm to be used for integration.

To change the default integration parameters for either algorithm, click on Tools-Settings – Quantitation Integration Algorithm and select Quant method editor settings.

Figure 38. ‘Analyst Classic’ Default Integration Parameters dialog.


Figure 39. ‘IntelliQuan’ Default Integration Parameters dialog.

The IntelliQuan algorithm uses one of two Peak-Finding Parameters, Automatic IQA or Specify Parameters MQII. After integration of peaks using the IntelliQuan algorithm, the user can choose which Peak-Finding Parameter best fits the data set. This is done in the displayed peak integration parameters in the Peak Review Pane or Window.

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13 Quantitation Wizard - University of Colorado Denver · 2010-07-29 · Quantitation Wizard 1 ... the “internal standard” name and its corresponding MRM transition of ... enter