Internal Standard Calculations for divided samples

[THNDRacket]

I'm asking for advice and submitting this to the Data Systems section because it deals with the calculation of internal standards in a sample that becomes divided. Feel free to move this if there is a better section for it.

Specifically, we are analyzing lavage samples that contain a wash buffer and suspended cellular matter. The sample prep itself is very simple. We centrifuge the lavage, and analyze the supernatant directly. The pellet is resuspended by treating with 3M urea and PBS and then analyzed. The final volume of both solutions is the same.

What we are finding is that upon applying the internal standard prior to centrifugation, most of the internal standard (a deuterated form of the component of interest) is in the supernatant. There is however a good amount of the component in the precipitate. Whether it is an issue of how much time the component and internal standard are in contact with the biological material, they do not seem to proportion equally between the two fractions.

Even if they did partition equally, I still argue with myself on how to calculate the concentration due to the internal standard being split between fractions. I'm thinking we would have to sum the corresponding areas in the two fractions and calculate the ratio then. Has anyone had similar experience that could offer some advice? Thanks!

[tom jupille]

Naively, I would think the answer would be to add the internal standard independently to both solutions *after* other processing rather than before. That way its concentration in both solutions is known, and you can use the relative response to calculate the concentration of analyte in each solution.

[THNDRacket]

I considered doing it that way, but it seemed to defeat the purpose of serving as a check of the sample processing. On the other hand, sample processing is minimal, so I guess that isn't too much of an issue. Thanks for the advice.

[James_Ball]

I considered doing it that way, but it seemed to defeat the purpose of serving as a check of the sample processing. On the other hand, sample processing is minimal, so I guess that isn't too much of an issue. Thanks for the advice.

I would use two monitoring compounds, one as internal standard added separately to each fraction before injection and another added before to demonstrate the partitioning into each fraction. The first would be used for accurate quantitation of components in each portion of the sample, the second gives you relative concentrations in each.

[lmh]

I think JamesBall is right, though if you want to be super-careful about it, even his two standards may not be enough. The situation is this:

The standard you add individually to the two subsamples is what you use as an internal standard in the chromatography software's quantification method, and it will take account of all losses and varying measurement efficiencies from the point you added it onwards. You can't really add this internal standard before dividing the sample, because to do the compensation for varying instrument efficiency, you need to know accurately how much internal standard was added to the sample you inject.

The remaining problem is whether you lost material before you added this internal standard (I don't think knowing the relative concentrations in each fraction is terribly important since you get that as a byproduct of knowing the absolute concentrations in both fractions). To be honest, I can't see how you could lose material before the split, but if this is a possibility that you need to test, then the correct thing to do is add the second standard, as JamesBall suggests. The problem is that this standard is also subject to varying instrument efficiency, so it won't necessarily be quantified accurately. For that reason, it needs its own internal standard, perversely added later! I think this is all getting too over-cautious, but the absolutely right way to do it, I think, would be to have two different isotopically-labelled standards, one added before the split, the other after. The one added after acts as true internal standard for both, and the one added before acts as a QC sample. If you added 10 units of the "add-before" standard, and you get back 3 and 4 in the two fractions, then you know you've lost another 3 somewhere during sample extraction. It would take some fast talking to persuade me to get two isotopically-labelled internal standards though. Usually I'm lucky to get just one.

[James_Ball]

It would take some fast talking to persuade me to get two isotopically-labelled internal standards though. Usually I'm lucky to get just one.

There are some that you can get that are fairly inexpensive such as the ones used as internal standards and surrogates in EPA methods like 8260/8270 that come as a known concentration is methanol/acetone/methylene chloride. If that would work, it would be less expensive than trying to source a pure labeled compound.