The Great Internal Standard Debate

[Toxtech]

Hi Everyone,

We have a method consisting of over 75 analytes and we are wondering how many Internal standards to run? Ideally we would run 1 for each analyte we know, however with the method as large as it is doubling the amount of compounds seems like a bad idea. Before you tell us to make it smaller, we have. We used to have one method, we now have 4. Our samples do not undergo any prep except dilutions. We are using some for calculation, and others to monitor injection. Any advice would be appreciated.


Thanks

[Peter Apps]

If the method is dilute and shoot, why do you need internal standards - what are they correcting for ?

Peter

[James_Ball]

If the method is dilute and shoot, why do you need internal standards - what are they correcting for ?

Peter

Even with this type of injection it is good to have internal standards to monitor and compensate for any changes in the injector or detector sensitivity. If the injection port gets dirty during analysis of samples it is good to know as you progress through the run and most methods will allow you to use the internal standards to compensate for some loss of sensitivity before needing to recalibrate.

As for number of internal standards to use. I have about 100 compounds in my volatiles analysis method and I use 4 internal standards and 4 monitoring compounds, internals to calculate from and monitoring compounds to monitor if overall response factors are changing. Example, use Chlorobenzene-d5 as internal standard to quantitate with, and use 4-Bromofluorobenzene to monitor how compounds are responding relative to the Chlorobenzene-d5, if the monitoring compound drifts you can assume other compounds are drifting as well.

[Peter Apps]

Hi James

I wasn't suggesting that the OP did not need internal standards (though that is a possibility), just trying to find out what the standards were correcting for. If it is e.g. inlet discrimination or inter-injection variation in transfer of analytes to column, then one low MW IS at the beginning and one heavy one at the end will probably cover it. If there is progressive loss of particular analytes as the inlet gets dirty then you have to match chemistry between IS and analyte. If detector drift is the problem then you need an IS that mimics the fragmentation of the analyte (assuming MS which is default for these multi-target methods).

The one thing that the IS does not need to correct for is sample prep recovery.

Peter

[MSCHemist]

As was mentioned it depends on what the ITSD is correcting for. If it is just instrument variations just one ITSD should suffice. If you have extractions, derivatizations, SPE's concentrations etc than you need and ITSD to behave chemically as your analytes so just one might not cut it.