Choosing an Internal Standard

Adding an internal standard (IS) to an assay can be an excellent way to often improve method precision and accuracy. An IS can account for signal suppression (or enhancement), that may be caused by the sample matrix. When using an IS, the response of your target compound(s) is compared to the response of the IS. The internal standard is added at a constant amount to all standards, controls, and samples. Since it is a constant, the IS can be monitored to determine if an individual injection is valid. Additionally, the IS can be used to calculate relative retention time and assist with peak identification.

Choosing a proper internal standard is the key to the success of using one. The IS should be chemically similar to your target compounds, and it should not be present in your sample(s). I would recommend reviewing your target compound’s structure and boiling point and choose an IS that would be similar. Ideally, one would have an IS for each target compound in one’s method, but this can become impractical with large compound lists. In these cases, choose a handful of compounds to match early eluting, mid-eluting, and late eluting compounds in your assay.

If you are using a mass spec detector, an isotopically labeled analog of your target compound makes an excellent IS. These are compounds that are deuterium labeled (contain deuterium in place of hydrogen) or 13C labeled (contain 13C atom instead of 12C atom). An example would be using Atrazine-d5 as an IS for Atrazine or other triazine herbicide compounds. Isotopically labeled analogs will chromatograph extremely similar to their non-labelled counterparts.

The drawback to isotopically labeled internal standards is that they are not available for every compound. Additionally, they coelute with the non-labelled target compound; hence, they can only be used with mass spectrometry. With a GC detector, such as an FID, ECD, or NPD or LC detector, such as UV or Fluorescence, these coelutions will lead to headaches in the lab. In these cases, one needs to look at for an internal standard that is similar to the target compound but not found in your sample. Many environmental assays use an internal standard that contains bromine or fluorine when analyzing chlorine containing compounds. As an example, a compound such as 4-bromofluorobenzene could be a suggested IS for chlorobenzene analysis. In the case of medical cannabis potency testing, my colleague Jack Cochran used compounds somewhat similar in structure and behavior to the cannabinoids that were present.

There are a few other Restek blog posts that discuss the use of internal standards for specific assays:

Great Internal Standards for Pesticide Residues….SRMs too!
TO-15 ISTDs – What do they mean to you?

Next time you are doing method development, consider adding an internal standard to your assay. You should end up with improved precision and accuracy, along with a more robust method. By reviewing your targeted compounds chemical properties, you will be on your way to choosing an appropriate IS.

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