Chromatography Forum

I am developing an HPLC method. It works well for my samples except one matrix spike giving me low recovery. I am thinking about using an internal standard to offset this issue; however, I hesitate to do so because I have to deal with chromatography issue again (resolution may be a problem). I have seen some people using standard preparation- prepare standards and samples in the same way (i.e. both through extraction procedure) for theire methods. Is it a good approach to sovle low recovery problem?
Thank you very much.
John

If it is truely low recovery, then the method needs improvement, and it is very difficult to "fix" the problem with an equally ineffective sample preparation method using a standard.

If the method is a LC/MS method, the problem could be ion suppression, in which case a better sample preparation technique is again the best solution.

Thank you, Uwe! It seems to be a fairly common misconception that it is easier to adjust an analytical method to compensate for an inadequate sample preparation technique than to improve the sample preparation. Your reply was very concise and accurate.

I fully agree. I do need to improve the sample prep procedure. Is it correct that standard should be never through sample prep?

That may be necessary. First I would see if there are alternate procedures in the literature which may give less matrix effect. If there is not an acceptable alternate you may be able to modify your current prep to decrease matrix effects. In my opinion running your standards through the sample prep should be used only when there is no other way to remove the matrix effects.

Good luck.

One can run into quite a dilemma in regard to recovery. If you do a very robust precleaning (for instance, an extraction with more than enough solvent to get ~100% recovery) you may get lots of the matrix, which can interfere severely. If you make a compromise betweenn matrix extraction and recovery of analyte, you will most likely have trouble in reproducing the recovery (you better make sure that no parameters are changed or changing).
As mentioned before, I think that quantitative work is best done with a combination of internal (added at the beginning of the workup) and external standard. That way you avoid the negative aspects of internal standards, but have all its positive benefits like knowing immediatly (in every analyzed sample) when something didn´t behave.

A guideline has been published by Matushewski et al in Anal Chem in July last year on how to deal with such situations, especially with LC/MS methods from bodyfluids.

Uwe,

Perhaps there is an error in the reference you mentioned above. There is no Matushievski (or similar) in the Anal. Chem 2004 author index.

I found some names like Mathiessen, and Matoussi, Matsumura, etc, but their publicactions do not seem to be related to this subject.

Perhaps you can clarify the reference.

Benjamin

Time flies:

Matuzewki, B. K.; Constanzer, M. L.; Chavez-Eng, C. M., Anal Chem. 75(13) (2003), 3019-3030