From my background reading it is suggested you use internal standards in each LC-MS sample in order to improve the quality of data analysis. However, one of the examples given is of ~8 isotopic compounds such as L-lysine d4 of which many are very expensive and others do not mention what the internal standards are. Is there a minimum number of standards required for a robust experiment or are they specifically dependent on the experiment itself? I currently use pooled QC samples and use reference compounds to calibrate the machine but nothing else.
Any idea/advice would be great,
Niall
If you want to do real quantification then you need an internal standard for each compound you want to measure.
A weaker approach would be external calibration curves with non-labelled standards. This would not take into account the matrix effect. So unless you can validate that this makes some sense with your particular matrix by comparing to an approach with labelled standards first I would not give any validity to such an approach.
For untargeted metabolomics people disagree heavily. Some use a single standard, some try to use a standard for each compound group or possibly group by retention time.
I have yet to see anyone show that internal standards help anything in untargeted studies...
EDIT: I should clarify that my comments regarding untargeted was for the idea that you can use internal standards to quantify in an untargeted setting and the idea that you can use internal standards to correct analytical drifts.
As
@stacey.reinke and
@romanas chaleckis pointed out they can however be very useful to check the system.
Thanks Jan,
I was under the same impression for untargeted. Some reported using really expensive isotopes for each group (up to £300 for 20mg) and I don't see how it added to the analysis?
I have also considered using a chromatography check solution at the start and the end of each run to check that this is taking place properly, is this overkill taking into account I will be putting multiple pooled QC's throughout?
Thanks again,
Niall
Hi Niall,
In the labs I've worked in, we've generally added a mix of 6-8 internal standards for untargeted LC-MS; these have often spanned the range of chromatographic retention and mass ranges. We used them to evaluate retention time stability, mass accuracy, changes in instrument performance over time (i.e. loss of signal), and matrix effect.
If you are in Manchester, you could go speak with the metabolomics folks at Manchester University. They have a lot of experience in mass spec and could give you some good advice.
Cheers,
Stacey
Hi Niall,
I think for quantification with expensive isotopes you are much better off with targeted methods. For untargeted work we use internal standards for technical check (as Stacey suggests). In that case, depending on chromatography and matrix, cheap exogenous compounds not expected in samples can be used- e.g. Good's buffers for HILIC, drugs and pesticides for RP.
Cheers,
romas