How to Average Calibration Responses in MassHunter Quantitative Analysis

Created by Xu Li-hua 7 months ago | Last modified by Carlos Vargas 1 month ago

This Information Applies To: Agilent MassHunter Quantitative Analysis Software

Issue

When creating a MassHunter Quantitative Analysis Batch with many samples, several sets of calibration standards may be included. If your protocol requires the use of the average response from several sets of calibration standards to define the calibration curve, this article demonstrates the steps needed to perform this operation in MassHunter Quantitative Analysis.

Steps to Follow

The steps below are demonstrated in MassHunter Quantitative Analysis version 10.0.

1. Initially create a calibration table based on all the sets of calibration standards for all concentration levels in the Batch (see Figure 1).

Figure 1. View > Batch Table Layout > Flat Table

2. Click Analyze Batch to process the batch and populate the calibration table with the calibration responses. The Calibration Curve window will now show the number of Levels and Points currently in use. In this example, 7 Levels and 14 Points (two Points per Level) based on the multiple levels and points as defined in the Batch Table (see Figure 2).

Figure 2. The calibration curve window of 7 levels and 14 points

3. Edit the Method – Method > Edit or F10 (see Figure 3).

Figure 3. Method > Edit

4. Click Method Tasks > Method Setup Tasks > Concentration Setup. Verify for each compound that all the calibration sets levels and responses are populated in the Method Table (Figure 4).

Note: If required the concentration values (Conc.) can be reviewed and adjusted for situations where a compound does not have the same concentration as the value set for Expected Concentration (Exp. Conc.) in the Batch Table.

Figure 4. Concentration setup table

5. From the Method Edit view, click Method > Average Calibration Replicates (see Figure 5).

Figure 5. Average Calibration Replicates option

6. Select All or click the individual compounds to be calibration averaged (see Figure 6).

Note: If the quantitation method uses internal standards, then all the compounds associated with a given internal standard MUST be selected.

Figure 6. Compounds selection dialogue box for average calibration

7. Review the Concentration Setup table, there will now be a single level per concentration instead of the previous multiple levels per concentration (see Figure 7).

Figure 7. Concentration setup table

8. Select Exit in the Method Setup Tasks and when Apply Method dialogue box is displayed, select Quantitate and then click Yes (see Figure 8).

Note: Do NOT select Analyze - Selecting Analyze will recreate a new calibration curve based on the sets of calibration standards and concentration levels defined in the Batch Table.

Figure 8. The Apply Method dialogue box

9. The Calibration Curve window will now show the number of Levels and Points currently in use after the Average Calibration Replicates step. In this example, there are now seven Levels and seven Points (one Point per Level) (see Figure 9).

Figure 9. One point per level in the calibration curve window after Average calibration replicates step

10. Only reprocess the batch using Quantitate Batch – avoid reprocessing using Analyze Batch as this will recreate a new calibration curve based on the sets of calibration standards and concentration levels defined in the Batch Table (see Figure 10).

Quantitate Batch will retain the current Average Calibration Replicates… single level per concentration and process all samples using that protocol.

Figure 10. Quantitate batch option