Internal standard in LC-MS/MS

[smartin]

Hi,

One basic but important issue about I’m not really sure….
When you work with MS/MS (in my case, LC-MS/MS), it is preferable to use deuterated for internal standards.
But, if I’m developing a method for the siMultaneous quantification of more than 1 analyte, have I to use the specific deuterated as IS for everyone? Or can I use only one for all of them?
That’s, analyte 1, analyte2, analyte3, and I use analyte1-d8 as IS for all of them. Is it correct?
Or I should use analyte1-d8 for analyte1; analyte2-d8 for analyte2; analyte-d8 for analyte3?

Thank you very much!!!!

[vahur]

Hi,

One basic but important issue about I’m not really sure….
When you work with MS/MS (in my case, LC-MS/MS), it is preferable to use deuterated for internal standards.
But, if I’m developing a method for the siMultaneous quantification of more than 1 analyte, have I to use the specific deuterated as IS for everyone? Or can I use only one for all of them?
That’s, analyte 1, analyte2, analyte3, and I use analyte1-d8 as IS for all of them. Is it correct?
Or I should use analyte1-d8 for analyte1; analyte2-d8 for analyte2; analyte-d8 for analyte3?

Thank you very much!!!!

You should have internal standard for every analyte seperatelly. Even if one takes into account all the sample preparation recoveries the matrix effect, especially if using ESI, can be very different at your retention times and you could not take it into account with one internal standard.

[lmh]

Yes, ideally you should have each compound present as a deuterated thing for the reasons vahur explained, but no correction is perfect.

You need to think about what might be going wrong.
(1) You might have chemicals coeluting with your analyte, that disturb its ionisation. If so the ionisation efficiency in the sample will not be the same as in the standard. This is where deuterated internal standards are best because they also coelute with the problem (a standard that elutes a minute away from the interfering chemical will tell you nothing). They will also suffer in the same way as the analyte, because they are chemically nearly identical, so their ionisation efficiency should be the same as that of the analyte.
(2) You might have general dirtiness of instrument increasing with time, in which case lots of analytes will suffer from a similar drop in efficiency. There may be some dependence of this on chemical characteristics, but similar chemicals suffer similarly. To correct for this, you could use a single internal standard for all analytes, and although it would be an approximation, it might be better than nothing.
(3) You might be losing analytes during extraction. This might be very general (see 4 below) or it might be that you lose, say, acidic things. If so, an internal standard that is chemically similar to your analytes, but not identical, may be appropriate.
(4) You may have general losses and variations, such as samples where the solvent may have evaporated a bit, causing a volume change, or where you may have lost a bit of solution at some stage in preparation. In this case any internal standard will do, even one chemically different to the analytes.

Depending on the environment in which you work (level of quality control needed), you may not always need the best internal standards. In particular, if you are working in an academic environment without quality regulation, and you're working on a novel compound for which deuterated standards aren't available, you will just have to do the best you can, and most good reviewers will understand this.

[Arne]

Three things:

The very best internal standard would contain carbon 13 because deuterium does induce a slight chromatographic shift - deuterated compounds elute earlier. I have recently read about this shift from a group that tried to do relative quantitation using mass tags that contained deuterium.

I would like to agree with the previous reply. Often it is not feasible to have an internal standard for every analyte. We have had quite good precision (rsd <3%) and accuracy (within 5% of known standard values) using no internal standards at all but only an external calibration curve.

Even if you only have one internal standard (such as C17 FAME for fatty acid profiling in GCMS) that will correct for injection volume variation and general instrumental drift.