MassHunter Quant: Reference Windows, RT Window

In Quant, there are at least 3 retention time related settings: Left/Right RT delta in RT setup, then Non Reference window and Reference window in Globals setup.

When using only the RT deltas to review integrated data in the compound information pane, using narrow RT deltas can make the compound view narrow and can leave peaks distorted. For some non-MS data, it can be helpful to see a wider range (ex. window = 1 min). This can be achieved by increasing the RT deltas.

However, when increasing the RT deltas, the integrator might select the wrong peak: ex., integration for compounds eluting adjacent to a surrogate may incorrectly select the surrogate, requiring manual intervention. In this case, tightening the deltas is not a suitable solution. After some experimentation, the integrator does appear to become more selective by decreasing the reference window % values rather than RT deltas, and seems to increasingly integrate peaks that more closely align with the compound's set retention time (which is good).

I would like to understand this behavior better to set up MassHunter to allow for a broader view of the chromatogram when scrolling through compounds in the compound information pane while still retaining selective integration based on RT for non-MS data.

Does anyone have any documentation or experience that can describe the functions and difference between "Reference Window" and "Non Reference" Windows relative to RT delta, and if this application is correct? As a bonus, there is even an RT window setting that I can't find documentation on either.

Thank you!

  • Hello  ,

    This is a common point of confusion and a very good question. The information is available within the help and the data set manuals, but it is really spread out and not easily pulled together, so let me try to explain.

    The Reference and Non Reference windows determine the RT range that a peak must be in to be considered the primary hit peak for a compound. The Reference window setting is only for ISTDs that are also flagged as Time Reference compounds for RT correction. Only ISTDs can be Reference compounds since Quant expects any Time Reference compound to be in every sample. All other compounds, targets, ISTDs, or otherwise, have their recognition windows set by the Non Reference Window. 

    By default both of these settings are very wide, up to 100 percent or more of the retention time. For most compounds this will be well outside of the Left and Right RT Delta window. If you enable the Reference RT and Recognition window markers in the Compound Information properties and then manually widen the RT display you can see the markers.

    So then what does the Left and Right RT Delta window do? These settings are used to determine the data extraction range for a given compound. It does not set the recognition window, but of course the recognition window will be within this range. This data extraction window will determine how much baseline information the integrator has to be able to find the start and end point of each peak. That is why peaks may not be integrated if the RT windows are set too small, since there will not enough information about the baseline for the integrator to be able to find a peak. However, as you note, as you make this wider you increase the possibility of other peaks being found that may be misidentified as your target compound, especially for less selective techniques like 2D, full scan, or SIM data. Though even accurate mass or MRM data may have issues as you widen this window.

    Having the correct peak consistently identified then comes down to having a wide enough RT window for consistent integration, and setting the recognition window narrow enough to find the correct primary hit peak for your target compound. Though you will need to be careful about setting the recognition window too narrow, because then slight shifts in RT or peak width will cause the compound not to be found. 

    Another setting that can help with consistent peak identification is Criteria. This column can be added to the method view and you can change it to Close RT or Close RT with Qualifiers. This can be particularly helpful if there are very large peaks close to a given target but the RT range can't be made more narrow due to lack of baseline information. 

    In the latest 10.x versions there is also a Reference and Non Reference Window Override column that can be used to set the recognition window on a compound by compound basis. This can be useful for very long runs where the late eluting peaks are much wider than early peaks.

    As for the RT Window column, that is actually the outlier setting for the retention time, 10.0% by default I believe. I agree the column name by itself is not very helpful, and of course since that is a common term, finding info on that column in the help is a bit of a challenge. 

  • Howard,

    Thank you for the detailed reply! This will be a great reference for future method development - having the full story of how these parameters contributes to consistent integration/identification is extremely helpful.

    So, for our goal then of having a wide RT window (to improve the review of some short/wide peaks) while retaining integrator selectivity, we should increase our RT delta range and decrease the Non-reference window. Since our retention times are relatively consistent (and are particularly important for our 2D data), decreasing the RT Window from the default of 10% to ~2% should help review by flagging misidentified peaks as RT outliers.

    Let me know if I got any of that wrong, and thank you again!

  •  ,

    Depending on your chromatography it can be a bit of a balancing act, but generally, yes, open up the RT window a little and tighten up the Non Reference Window. For 2D data you may also want to try setting the Criteria to Close RT.

    As for the RT Window outlier just make certain it is set narrower than your recognition window. Otherwise peaks will never get flagged for RT. 

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