Stable Isotope Internal Standards for LC-MS/MS

I have been doing LC-MS/MS method development for almost 15 years and have used stable isotope internal standards for much of that time. Recently I have been asked to start to tutor other staff in my group on quantitative LC-MS/MS method development. I have been trying to find references for the use of stable isotope internal standards but have been unsuccessful. Does anyone know where I can find references for a detailed explanation of the calculations, consideration and propagation of errors that occur with the use of stable isotope internal standards? Specifically I am looking for information regarding internal standard recovery limits and the propagation of error if the internal standard recovery is too low or too high, impact of density on the calculation (standards by volume but samples by weight), limitations for correcting matrix effects, and limits for using the internal standard to correct for sample dilution or concentration during sample preparation.


  • Hello:

    One of the Waters Senior Applications Chemists in Chemistry Applied Technology wrote a document a while back that describes some of the limitations of stable isotope labeled internal standards when it comes to compensating for matrix effects. It has some references in it as well- again describing instances where SIL internal standards do not adequately compensate for matrix effects.

    You can download the paper here:

    The literature code is 720002438en if that helps.

    Hope this helps.

  • This provides some of the information. But for the most part the referenced articles are experimental discussions as to why dueterated compounds may or may not be suitable internal standards. While this has helped identify a few additional sources of error for a couple of the methods I am working on it does not address the more fundamental questions around the calculations of the internal standards and the errors that occur due to the partial recovery of the internal standard. What are the practical limits on using the internal standard to correct for sample losses and matrix effects? Can an internal standard be used to correct for a 95% loss of analyte during sample preparation without introducing significant and unrecognized error? How would I experimentally verify that the recovery (both low and over recovery) of the native compound is not incorrectly adjusted. Does the density of the solution impact the calculations? Can standards be prepared based on volume but sample prepared based on weight without a density conversion as the IS is added based on a final volume concentration or does this introduce additional error in the data manipulation.

    Any and all thoughts are appreciated.


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