Modify the Integration Parameters in MassHunter Quantitative Analysis

Answer

This Information Applies To: Agilent MassHunter Quantitative Analysis Software

Issue

Chromatographic peak integration is a critical part of accurate and reproducible quantitative data analysis. The integrator type and the integration parameters can be adjusted to optimize peak integration. 

Available Integrators

MassHunter Quantitative Analysis has three categories of integrators, with a total of seven automated integrator types. The results from all integrators can be filtered with the Peak Filter tab. The same integrator type and peak filters are applied to both quantifier and qualifier signals by default.

1. Agile, Agile2, MS-MS, and MS-MS(GC) are parameter-less integrators that determine the best approach to integration based on the data being integrated. 
   1. Agile – Second-generation parameter-less integrator intended for MS data types (MassHunter Quant default).
   2. Agile2 – Third-generation parameter-less integrator with improved baselines and higher sensitivity to smaller peaks – recommended for GC/MS data types.
   3. MS-MS and MS-MS (GC) – First-generation parameter-less integrator intended for MS-MS data types, not recommended for MS data types and requires a minimum of 64 data points in the chromatograms.
2. General and Universal offer compatibility with previous Agilent integrators in the MSD ChemStation data analysis software.
   1. General – Compatible parameters to the MSD ChemStation RTE integrator.
   2. Universal – Compatible parameters to the MSD ChemStation Genie integrator, which supports timed events and, can be selected when integration event control is required over user-specified time ranges.
3. Spectrum Summation is a specialized integrator that enables area summing, which doesn’t, require chromatographic peak detection.
   1. Spectrum Summation – Integrator designed for chromatograms with poorly separated peaks, irregular peak shape, etc. Sums the signal at each data point across a time range, excludes the signal below a user-defined baseline offset. The baseline is always horizontal and the reported RT is the center of the time range.

Tips:  For most GC/MS applications, it is recommended to initially use the Agile2 parameter-less integrator and filter the peak integration results using the Peak Filter tab. To reduce false negatives: Do not use peak filter on Peak Area % or Peak Height % for complex sample chromatogram quantitation. These types of samples may have other large peaks in the EIC signal that can filter out a smaller target compound response and generate a false negative result. The default Peak Filter is >= 5% Area. Agilent recommends using an absolute Peak Area or Peak Height based on the signals from the lowest calibration standards or the required reporting limit. There is an easy-to-use Tool to set Peak Area relative to each compound's lowest calibration standard for all the compounds from the Custom Actions – click Method > Edit > Tools > Actions > Set Peak Area Threshold – typically set to 50 or 25%. This will significantly reduce both False Positives from Quantifier and Qualifier peak areas less than the lowest calibration standard and False Negatives from using the default >=5% of Peak Area within the EIC window.

Steps to follow

1. From the Batch Table view click Method > Edit to open the Method Tasks, click Advanced Tasks > Integration Parameters Setup. In the Method Table tab, select Quantifier > Int. to open the Integration dialog box for that compound and view its Integrator type and settings (Figure 1).

   
   Figure 1. Integration parameters setup

2. In the Integration dialog box (Figure 1), Integrator tab select the Integrator type required. The default is Agile. If the integrator is a type requiring parameters (general, universal and spectrum summation) set the parameters:

   1. General – Click General tab (Figure 2) and specify the integrator parameters:

      Data point sampling:  Used in calculating the slope sensitivity. The default setting of 1 uses every data point, set to 2 for every second data point, set to 3 for every third, etc. Integration performance may be improved for broader peaks with many data points by adjusting this parameter. 

      Smoothing: May improve results for high noise data. It may also be useful anytime the integrator shows a tendency to split chromatographic peaks. 

      Detection filtering: For high noise chromatograms. Select a higher number of data points (3,5,7,9-point digital filtering) to reduce adverse integration from signal noise.

      Start and Stop threshold: Determine when a peak integration starts and ends. Increasing the stop threshold is useful to increase the amount of peak tailing integrated. 

      Peak location: Selects the retention time reported as the Top (Apex) or Centroid (center) of the peak.

      Baseline Reset: Controls resetting the baseline between peaks. Set the number of data points required to separate two adjacent chromatographic peaks. Set the

      If leading or trailing edge <, and Baseline preference: Adjust tangent skim baseline construction.

      
      Figure 2. Parameter setup for the General integrator

   2. Universal – Click Universal tab (Figure 3). The Universal integrator has Initial Parameters that remain in effect over the entire chromatogram unless superseded by a Timed Event. Set the parameters: 

      Threshold, Peak Width, and Area Reject: These settings determine sensitivity, baseline point, and small peak rejection respectively. Initial parameters cannot be deleted from the table, and are suitable for most applications. In more complex chromatograms and specialized applications, the use of timed event parameters enables optimization of the integration results. 

       
      Figure 3. Parameter setup for the Universal integrator

   3. Spectrum Summation – Click Spectrum Summation tab (Figure 4). The Spectrum Summation integrator requires settings for summing data points together over a set time range the horizontal baseline.

      
      Figure 4. Parameter setup for the Spectrum Summation integrator

3. Peak Filter – Click Peak Filter tab (Figure 5) and set the parameters, which apply to all integrator types. Peak Area (counts) or Peak Height (counts) are the most widely used. Peak Area (%) >= 5 % of the largest peak can cause false negative results in samples with a complex chromatogram as large EIC peaks cause smaller peaks to be filtered out.

   
   Figure 5. The Peak Filter tab

4. Apply the selected integrator type and peak filter settings to the current compound only (click Apply) or all compounds (click Apply to All). Click OK.

5. Qualifier table > the Int. Params. (Figure 6). The default option of Use same parameters as parent compound sets the same conditions as the quantifier and blocks other settings (Figure 7). The Peak Filters for the Qualifier can be set differently from the Quantifier when Use same parameters as parent compound is deselected (see Figure 8.).

    
   Figure 6. Integration parameter setup for the qualifier
    
   Figure 7. All tabs unavailable when Use same parameters as parent compound is selected (default)
    
   Figure 8. The Peak Filter tab is active when Use same parameters as parent compound is deselected

Learn how to effectively operate MassHunter software: Agilent 5977 GC/MSD with MassHunter Workstation e-learning path available from Agilent education GCMS-MH-2150scV3 - Agilent MassHunter Quantification for Single Quadrupole GC/MS with Cloud Laboratory (Version 10.1) e-learning course available from Agilent education