Quantify compounds using MS

If you want to quantify your samples using mass spectrometry, you must first find out which m/z values you can use to quantify the different compounds. Data Analysis helps you finding the appropriate masses and retention times.

Preparations

• To be able to carry out the procedure as described, you need the privileges Project Management > Edit content of project , Processing Method > Edit identification parameters and Data > Edit sample information . Privileges are configured in the Control Panel.

• If the project enforces method approval, the method status must be Generic. For details, see Method approval.

1. Load the data.

2. Make sure there is an MS method linked to the data (method configuration MS). Alternatively, create a new MS processing method and link it to your data:

   1. In the Processing ribbon tab, click New Method .
   2. Select the MS method configuration, and click Create method.
   3. Save the new processing method: In the Processing ribbon tab, click Save Method As .
   4. Link then new processing method to your injections: Select the relevant injections, and click Link Method in the Processing ribbon tab.

3. In the processing method, adjust the integration parameters. Consider using specific events for the extracted ion signals.

   1. In the Integration Events > Standard node of the processing method, select the Use signal specific events check boxes for the TIC SCAN, SIM, or EIC signals.
   2. Adjust the parameters for the relevant signals.

   For example, set Area reject to 200000 for TIC SCAN and to 10000 for EIC.

4. To extract ion chromatograms over a time window around each target peak rather than the entire run time: In the Extraction > Chromatogram node of the processing method, enter a value for the EIC extraction retention time window for target compounds.

   It is usually best to pick a time range that is wide enough to extend enough beyond the target peak to correctly assign the baseline points. For example, use an Absolute RT window of 0.1 min around each peak.

   With zero values, EICs are extracted over the full time range.

5. Configure background subtraction for MS spectra.

   In the Extraction > Spectrum node of the processing method, choose an appropriate Background mode for peak apex spectra.

6. In the Compounds > Identification node of the processing method, in the Qualifier Setup tab, check the settings for qualifier ions.

7. To get compound names automatically, based on an MS library search: In the Spectral Analysis > MS Library Search node of the method, choose an MS library from the drop-down list.

   NOTE

   If you want to use MS Library Search, you must install NIST MS Search and the required MS libraries first. NIST software can be acquired from Agilent.

   The status of the Automatically search TIC peaks check box is not relevant in this scenario.

8. Consider saving the method.

9. Reprocess the injection.

   All relevant peaks should now be integrated.

10. Right-click the injection in the injection tree, then select Add/Update MS compounds.

    OR: Alternatively, right-click the compound table or the chromatogram, and select Add/Update MS compounds from the respective context menus.

    The application automatically analyzes the data as described here, and adds the corresponding information to the processing method.

11. Name the compounds, and check and complete the compound table in the method.

12. In the Compounds > Calibration node of the method, provide the correct concentrations for your calibration levels.

    Consider saving the method.

13. In the Injection List window, provide the correct sample types and calibration levels.

14. Optional: To add compounds from other scan data to the same method:

    1. Open the other data file.
    2. Link the other injection to the same method.
    3. Select your original method. Then right-click the new injection, and select Add/Update MS compounds.

    The new compounds will be added to the selected method.

15. Reprocess the data.

    During reprocessing, an EIC for each m/z value (of both target and qualifier ions) will automatically be extracted and listed in the injection tree.

    The response of qualifier ions is compared to the response of the target ion. If it is within a defined response window, the qualifier is listed as Passed in the Injection Results.

    The EICs of the target ions are used for quantitation.