Integration | workflow LC
To calculate concentrations and evaluate the kit performance, Chromatographic peaks corresponding to each metabolite need to be identified and integrated. To perform the integration, upload or load MS data then define integration windows as described below.
Recommended workflow | adjust integration windows
- Perform auto peak picking
- Define integration windows for plate run
- Define integration windows for injections/wells
- Perform integrate plate run
Once integration windows are defined, an integration preview is shown.
Click Integrate. to integrate all peaks of the plate run.
The plate run integration status is shown in the Quantification workflow guidance.
Auto peak picking
After uploading MS data of a LC plate run (LC part of a kit), auto peak picking identifies the peaks and automatically sets the integration windows for each metabolite for the whole plate run. Two options are available.
| Auto peak picking for defaults | Auto peak picking |
|---|---|
For one plate run and for each metabolite, the same integration window is defined (default integration window).   | For each chromatogram and each metabolite, specific integration windows are defined.   |
| Recommended | Useful for special cases such as RT shifts or matrix effects occurred. |
Click Auto peak picking for defaults.
Auto peak picking is available for the following kits
If no default integration windows are available, auto peak picking for defaults is performed automatically when the data is uploaded.
Check integration windows
The quality of the integration windows defined by the auto peak picking is indicated next to the corresponding sample and metabolite. Check samples and metabolites with purple and red indicators.
For detailed integration window status information, refer to section Integration windows features.
Example for a QC2 sample with a purple indicator
- Select a well with a purple indicator.
- Select a metabolite with a purple indicator.
- Update the corresponding integration window by clicking once on either side of the peak. The indication will disappear (See example below)
When all integration windows of a sample are well defined,
Auto peak picking settings
The threshold required to highlight low confidence peaks from the auto peak picking can be defined in the settings.
Settings > Quantification > Integration
Low, medium, or high sensitivity can be selected to detect integration windows for improvements.
Example
After performing Auto peak picking, it is highly recommended to check all integration windows!
- Integration details can be found in the kit user manual in section Peak identification or in the Technical guide - RT adjustment document located in the kit files.
- For some kits (e.g. MxP Quant 1000), integration guidance screenshots are displayed alonside sample chromatograms in the Integration section.
- Follow the instructions described in the next section Integration windows for plate run.
Integration windows for plate run
After applying auto peak picking it is highly recommended to check all integration windows.
Define integration windows as described in the kit user manual section Peak identification or in the Technical guide - RT adjustment document located in the kit files.
Apply integration windows to plate run
Integration windows that have been applied to the plate run are indicated by the "default mode" status in the chromatogram window.
- Select all injections from the table Wells.
- Switch the zoom slider to All.
- Select the first metabolite from the table Metabolites, e.g. 1-Met-His-ISTD1.
In the Chromatograms panel, chromatograms from all injections of the selected metabolite are overlayed, e.g. 1-Met-His-ISTD1.
- Check the integration windows of all metabolites and internal standards (ISTDs), shown in the list Metabolites.
Defining integration windows for a plate run summary
- Select all injections from the table Wells.
- Switch the zoom slider to All.
- Define an integration window for the selected metabolite in the chromatogram, where is shown.
Default integration windows are displayed as solid lines, .
Integration windows for specific injections or wells
Specific integration windows for each metabolite can be defined for individual injections or wells.
Required when
- RT shifts occurred during data acquisition
- Signals close to a metabolite peak are detected (due to contaminations or matrix effects), e.g. "Asp".
- Select one or more injections or wells from the table Wells, e.g. sample "2022-12".
- Select a metabolite from the table Metabolites, e.g. "Asp"
- Adjust the integration window by clicking once on either side of the peak.
- In the view Chromatograms define a suitable integration type, Valley to valley or Horizontal.
Defining integration windows for specific injections or wells summary
- Select one injection/well from the table Wells.
- Select one metabolite from the table Metabolites.
- Define specific integration window.
Specific integration windows are displayed as dashed lines, .
Integrate LC plate run
After finalizing all integration changes, perform the integration procedure of the selected plate run by clicking Integrate.
The progress is indicated as a blue bar at the upper left edge of the screen.
For a more detailed look at all integration features, see LC integration features
Upload and integrate data of each plate run individually, e.g. for Quant 500 XL kit