Calibration standard added prior to cleanup step

[Pepter]

Hi guys,
Will it be mistake/error if i make calibration samples by adding calibration standard after the extraction prior to cleanup step (SPE cleanup).

[Don_Hilton]

You calibrate according to what you want to measure. (This sounds like you are making an in-matrix calibration?) So, do you want to report the quantity of material in the sample after extraction or as handed to you before extraction? If you can assume that extraction is 100% efficicient, it might be reasonable to add the standard after the extraction step - but there is this thing about assumptions... (Assumptions have a way of getting us in trouble.)

So, without further information - this is not sounding like a good idea.

Are you adding an internal standard? And, if so, where in the procedure?
Have you measured recovery through your method?

[Pepter]

Hello
Yes iam making in-matrix calibration and and deutered internal standard is used.

Extraction method in short:
1) Extraction with MeOH
2) Filtration
----------------
3) SPE cleanup
4) Evaporate step
5) Redissolved in mobile phase

For recovery samples i add standard as usual before extraction
The ISTD standard is added after filtration before the SPE cleanup i also make calibration samples from here.

Step 1 and 2 don't give any high losses in analytes, the problem is SPE and evaporation.

[Don_Hilton]

Step 1 and 2 don't give any high losses in analytes

It is the accuracy related to those small losses you lose.

Is there a reason not to add internal standard and calibration standards before extraction?

[Pepter]

Thank you Don for reply

May i ask a question ? What will be difference between recovery samples and those for calibration curve if
i done them in the same way ? Is it correct from the methodological point of view.

Results that i obtain said yes but... i get a question from my boss "why you didnt make calibration samples by adding proper volume of standard to blank at the
end of procedure"

p.s.
sry for my bad english

[Don_Hilton]

The numbers you report for a sample are related to the point(s) where you add standards. The accuracy of those results relate to how recovery issues acros the steps.

If you add an internal standard to the material you recieve in the laboratory, and that internal standard is lost in exactly the same way as the analyte you are measuring, the ratio will remain constant even with variable recovery. Isotope labeled internal standards are the best for matching losses exactly. (But we do want to keep these loses small - you note that deuterated standards do separate slightly from the native compunds on the GC, so there is a very small difference in chemistry.)

So if you add standards after all the extractions are done - you are showing the abiltiy to measure what is left at the end, not the ability to make a measurement that accounts for what was present at the start. Most people who submit samples want to know what is in the sample they deliver to the lab, as delivered.

[Pepter]

Thank You.
I think i understand the role and way the ISTD (especially the deutered one) works.
My main problem is the way i prepare calibration samples in this procedure
Is the point 1 wrong ?

My old post where i ask about it:
How should the standarisation (calibration) look correctly in this method ?

Calibration:
1) Add both standards deutered and "normal" before column cleanup procedure after the extraction (extraction with MeOH/filtration) ? <---- (i dont it in this way)
2) Add both standards deutered and "normal" into vial after the column cleanup procedure ? <---- i understand thats the most common way of doing calibration samples
3) Add deutered standard before the cleanup and "normal" into vial ?
4) Add both standards deutered and "normal" before extraction ? (the calibration samples and recovery done in the same way)

Fortification (recovery samples)
1) Add both standards deutered and "normal" before extraction

[Don_Hilton]

The best practice is to spike the samples with internal standard when placing a subsample into the container in which sample preparation will start. When making an in-matrix calibration, the analyte is spiked at the same time. For computing recovery, the analyte is spiked into the sample when it is subsampled and the internal standard is added after all clean-up steps are completed.

In the evaluation of individual steps for losses or recovery in the method, the analytes can be spiked into the samples before the given step and the internal standard at the end of the step, with all other sample preparation steps being completed.

[Pepter]

I'm really grateful Don for your help.
Iam looking now for some publications where calibrations where done in the same way as recovery samples.
Maybe You remember titles or authors so i can look for it ? I google, but most of the chromatographic publications about
pesticides determination in different matrixes describe calibrations prepare like in point 2.

[Don_Hilton]

You may find standards spiked at the end of preparation in a published paper. There are about three reasons. 1) there may be some particular difficulty that prevents the standards to be spiked earlier. 2) The analytical work or at least the spiking with standards is secondary to the key teaching of the paper - and while not great, the story is worth publishing. 3) The reviewers let it go past - and should not have.

If I get a paper to review and internal standards are included after preparation, more than likely I will require some discussion on that. Otherwise, the paper will have an item that must be addressed before I can reccomend it be accepted.