In MassHunter, is there a way to set a time reference compound without it also being the internal standard?

I want to be able to use a time reference compound to calculate RRTs, but without making internal standard corrections for the quantitation.  I can do this easily in Chemstations and in OpenLab but when I try in MassHunter, I get a validation error that states "Only ISTD compounds can be used as a time reference.  This seems like an odd limitiation.

  • Hello  ,

    I apologize for the delayed response.

    MassHunter does require that Time Reference compounds also be ISTDs. RT correction is done on a compound-by-compound basis using the associated ISTD if the flag is enabled.

    Relative Retention Times are calculated based off of the target's ISTD RT and do not require that the Time Reference flag be enabled. Here are the descriptions from the DataSet manual.


    TimeReferenceFlag has the value true if and only if this TargetCompound row represents a time reference compound. Only internal standard compounds can be designated as time reference compounds.

    If compound B is a time reference for compound A, then the Quantitation Engine will multiply the retention time of A’s peaks by the ratio of the actual-to-expected retention time of B’s primary hit peak.

    TimeReference allows a time correction for a particular target compound. The approach is useful in the case where the target compound’s retention time drifts and the related ISTD used as the time reference also experiences the same relative shift. As a result, the Quantitation Engine will look for this target compound in a shifted retention time.

    Note that the extract retention time window does not shift, just the retention time where the target is expected. With the Peak Selection criteria set to Closest Retention Time, this shift in the expected retention time can improved the peak selection, especially when another peak exists in the same extracted chromatogram.


    RelativeRetentionTime represents the ratio of the target compound’s retention time to the related ISTD’s retention time. RelativeRetentionTime is a value used in calculation of OutlierRelativeRetentionTime.

    There is not an easy way to utilize these features for ESTD methods. You could make a duplicate of your target, assign your RRT compound as its ISTD, and use this copy to only evaluate the RRT. While less than ideal I'm not aware of any other way to do this in Quant. 

  • Thank you for confirming what I had found.  I figured that there wasn't a way around the time reference/ISTD link.  Mostly, I am surprised that MassHunter is limited in a way that Chemstations/OpenLab are not.

Was this helpful?