The need for recovery study in isotope dilution technique

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

9 posts Page 1 of 1
Dear all,

I am recently reading about the journal paper applying isotope dilution technique (IDT) in pesticide residue analysis with LCMSMS.

From what am I understand, the use of IDT help to compensate or correct the error that occurred during extraction (assuming internal standard (IS) was added prior to sample extraction) and matrix effect.

However, I noted that several journal authors mentioned that they also carried out recovery study for the method they have developed. The recovery study was carried out by comparing the area ratio of analyte and IS for sample spiked before extraction to the same ratio for analyte and IS added to the extracted solution (analyte and IS added after extraction).

My confuse is if IDT corrected the error or problem with recovery, why did the authors still need to carry out recovery study? What actually the reason to conduct such study and does it mean if the recoveries are bad, the method developed is also bad?

Hope to get explanations from peoples here who has experiences in using IDT.

Thank you.
Kenny
By doing the recovery study they produced evidence that the internal standard really did behave in the same way as the target analyte, rather than just assuming that it did.

Peter
Peter Apps
Peter Apps wrote:
By doing the recovery study they produced evidence that the internal standard really did behave in the same way as the target analyte, rather than just assuming that it did.

Peter


Thank you Peter,

Can you please elaborate more how recovery study can help to proof that internal standard behave the same way the target analyte does? Do you mean if the recovery perdentage is low, IST does not behave the same way as the analyte?

Thank you in advance for your explanations.
Kenny
Yes, that is what I mean. If the recovery is bigger or smaller than 100% then the IS is being extracted from the spiked sample differently to the analyte. It is possible to correct for the difference using the measured recovery.

"Recovery" is not the best word to use for this - "selectivity" might be better; denoting the degree to which the extraction is selective for IS or analyte.

Peter
Peter Apps
Thank you Peter. In one of the papers I read recently, the author was comparing peak area for the analyte of interest getting from injecting of sample after extraction and clean-up to the peak area of the analyte of interest in sample spiked after extraction and clean-up steps.

The author termed this comparison as recovery, my confusion is how this "recovery" is useful in the analysis if the internal standard and analyte of interest behave the same or very similar (example analyte of interest = methamidophos and IS= Methamidophos-d6) during extraction and clean-up steps?
Kenny
along wrote:
Thank you Peter. In one of the papers I read recently, the author was comparing peak area for the analyte of interest getting from injecting of sample after extraction and clean-up to the peak area of the analyte of interest in sample spiked after extraction and clean-up steps.

The author termed this comparison as recovery, my confusion is how this "recovery" is useful in the analysis if the internal standard and analyte of interest behave the same or very similar (example analyte of interest = methamidophos and IS= Methamidophos-d6) during extraction and clean-up steps?


Once again the critical question is whether the IS and the analyte really do behave the same. You must not simply assume that they do.

Unless I read the paper myself (which I do not have time to do) I cannot say why the authors may have done any particular thing in any particular way. The very best source for that information is the authors of the paper.

Peter
Peter Apps
One reason for the recovery study can be to determine what the actual sensitivity of the analysis method would be. If the instrument can measure 100ppm but you can only extract 10% of the target analyte from a matrix, then you can only accurately measure down to 1000ppm in the target matrix. Anything less than 1000ppm may not give you enough of the analyte in solution to detect it as most is retained in the matrix.

Recovery studies are a way to validate the method of analysis and to prove what the true limits of the method are in relation to the matrix and analyte being studied.
The past is there to guide us into the future, not to dwell in.
It's a real pity they've referred to this as a "recovery", because the way it's described (adding IS and analyte after extraction) it isn't. As Peter wrote, it's a test of selectivity.

With an internal standard method, you're assuming that the IS corrects for losses because if 33% of the internal standard has gone, you can be utterly sure that 33% of the analyte has gone. By spiking a blank sample with IS and analyte before extraction, and comparing the peak size ratio with results on spiking with both after extraction, you are checking whether the loss was equal. 33% divided by 33% is 1, 100%.

You can also make an estimate of what I'd regard as the real recovery, by starting with a blank sample, spiking with a known amount, and adding the internal standard (to correct for measurement-efficiency rather than extraction efficiency) after extraction. Then compare the amount of spike that you get back at the end with the amount of spike you know you added. This is also a very valid control as James wrote.

It gives you a sort of window of reliability (particularly valuable if you haven't done the selectivity test of adding IS+spike before and after). If you get back 100% of your analyte, then from the point of view of extraction, although the internal standard isn't doing you much good (there's no error to correct), it isn't doing you much harm either, because it's not actually making any correction. If the recovery is only 3% then alarm-bells should ring. The internal standard is now making an enormous correction. A tiny absolute error in the internal standard (say you get a 2% recovery instead of a 3% recovery) can make a huge proportional error in the final measurement. Also, of course, you've lost a lot of sensitivity, and it might be a sign that your extraction procedure isn't doing what you think (for example, if you've got a spinning-out-rubbish stage, or a two-phase separation, it could be that the analyte is in the wrong phase, or spinning out with the rubbish).

So in an ideal world, I'd do both: test the selectivity, and check the overall absolute recovery.
Thank you everyone for the explanations.
Kenny
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