There have been discussions on GC Message Board on the selection of ISTDs for GC and GC-MS. It is my understanding that the best ISTD candidate for LC-MS would be isotope-labelled analytes. What to choose for LC-MS if isotope-labelled analytes are not available? What would be good candidates for HPLC? Isotope-labelled analytes probably can not be used because they have the same RT. Any suggestions are highly appreciated.

Isotope-labelled analytes make good LC-MS standards *because* they have the same retention time (the MS can sort them out because of the MW difference). The ideal internal standard will have the same behavior as your analyte.

The exact choice depends on the chemistry of your analyte and the matrix/interferences but to my mind, the choices would be (in order of decreasing desirability):
- isotope-labelled analyte
- analyte homolog (e.g., with an ethyl instead of a methyl group somewhere)
- analyte derivative (e.g., an ester, etc.)
- analyte analog (i.e., many structural features in common)
- anything you can find that elutes near the analyte.

You may be surprised at how many isotope labeled compounds are available. Contact a company called Cambridge Isotope Laboratories, Inc. (Andover, MA) If they don't have it in the catalog, they might be able to make it for you.

I would add to Tom's sage advice the following. There should be a reason for using an internal standard. For example, you have determined that the detector drifts, there are work up losses, compensation for volume changes, what ever. When you understand what problem you want to fix with an internal standard, then selecting one becomes less a guess. So for example, if the problem is drift in response factor in MS, something deuterated is perfect. A chemically similar isomer may be next best, and so forth. On the other hand, if the internal standard is added to a work up to compensate for volume changes, then about anything may work.

Having selected a candidate for an internal standard, demonstrate that it helps by calculating results with and without it. I recently worked with a group that was doing carbohydrate analysis with pulsed amperometric detection. They used an internal standard with the hope that it would compensate for detector drift. When we independently plotted response factors for the analyte and internal standard they were not strongly correlated, so the internal standard selected was not adequately compensating for drift in analyte response factor. In another case, an internal standard was used to compensate for decomposition in a sample preparation step. When checked it was found the decomposition rates for the two compounds differed by a factor of 3. The moral of the stories, after selecting a candidate internal standard using you best chemical intuition, check to see that it is doing its job.

There is a price to pay for using an internal standard. Two peaks are measured in an internal standard analysis. Measuring two peaks doubles the chances for an interference, a not insignificant possibility in a complex misture, and it degrades the pecision by a factor of 1.4 (square root of 2) relative to an equivalent external standard method.



If you can't show you need an internal standard, don't use one. If