Use of Internal Standard

Harald Schoeny , October 02 2018
Harald Schoeny
I would like to know how you use your internal standard. As far as I know, you can either use it for an internal standardization of an external calibration curve (calibration curve produced with area ratio and concentration of the external standards, area ratio of sample is calculated back to concentration by using the slope and interception) or you apply an one-point calibration (use the known concentration of your internal standard in the sample of interest, conc. [analyte]= area ratio [analyte/ISTD]* conc. [ISTD]).
If you use the one- point calibration, do you use external standards to verify your method or do you just use them to have a level 1 identification?
Is it necessary to correct the intensity if the analyte differ in mass, number of carbons or number of double bonds? If yes, how is that done? How should the applied method reported in a publication and is it not necessary to do so?
I think it should be more clarified how an internal standard should be used and what limits a method has. The paper of Miao Wang and Xianlin Han (Mass Spectrom Rev. 2017 November ; 36(6): 693–714. doi:10.1002/mas.21492.) is very informative but I would be happy if more guidelines exist how to do it.
Thank you in advance, Harald Schoeny, University of Vienna
Alan Maschek
Are you asking about targeted or untargeted lipidomics?
We use single-point calibrations and we have spiked in other lipids to verify that our findings are accurate for our targeted platform. In untargeted analysis we are primarily concerned with relative differences between experimental groups and believe that reporting concentrations adds little value since ionization strengths and retention times between the IS and targets will be quite different.
If there is a mechanism to correct for the differences in number of carbons and/or double bonds then I have not seen it. Perhaps you should just report these values as semi-quantitative when using an IS that differs from the target, either in carbons:double bonds and retention times.
-Alan Maschek, Univ of Utah
Gerhard Liebisch
Dear Harald,

In principle both methods, application of calibration lines and use of single standards may generate accurate values.

Starting with an instrument I would recommend generation of calibration lines to get a feeling for the linear response of your instrument (this
should be always kept in mind when “real samples” are analyzed). Quantification based on a single standard may fail when significant differences in the individual analytical response are observed e.g. as for cholesteryl ester (DOI: 10.1021/acs.analchem.8b05013). In such a case either species response need to be corrected by response factors or individual calibration lines. When individual species response does not depend on structural features like double bonds or chain length, quantification by application of a single standard (of the same lipid class) delivers accurate values. Using a single standard, chain length needs
to be considered due to isotope abundance (type I isotope effect).

For application of internal standards we suggest the following: 

  1. Not present in the samples
  2. Addition prior lipid extraction
  3. At least one per lipid class (as defined by LIPID MAPS classification) is desirable
  4. High structural similarity to analytes
  5. Similar number of carbons
  6. Similar number of double bonds
  7. Simultaneous ionization with the analyte is desired
  8. Structural identical stable isotope labelled standards are considered as gold standard
Gerhard, University Hospital Regensburg, Germany
Rodolfo Dantas
Hello Dear,
I would like to know about the use of standards in shotgun lipidomics. Considering that I need to make a study based high-resolution mass spectrometry direct infusion (Q-TOF), and this analysis is just about fingerprinting between two cultures of cells.
1 - Do I need to add standards in my samples (ever)?
2 - If in this analyses I don't add a standard, my identification stay compromised?
3- Is it possible to consider it as qualitative analysis and carry out a potential identification?