Internal standards use!

[an_k]

Hello,

I have some questions about the use of internal standards in assay determinations. If I understand correctly, we use them when the analytical method (hplc method) lacks sensitivity. So with the presence of an internal standard and RRF use/correction we get the correct signal/concentration correlation and then we can determine assay values, right?
Could someone provide a practical example for this? I cannot completely understand it only on theoretical basis.

Thank you!

[DR]

Internal standards are used when some sort of extraction and/or derivitization steps are executed to increase sensitivity. Their use prevents bias (usually negative bias) in the results as a result of analyte lost during extraction and/or incomplete derivitization of the analyte. This is why it os important to choose an internal standard carefully. You want one that will be about as easy to lose and as hard to derivitize as your compound of interest is.

The ratio of analyte to internal standard area (analyte area/i-std area) for each injection is treated as the response for the compound of interest and pushed through normal external standard calculations.

[Consumer Products Guy]

We currently do not use internal standards in any of our HPLC procedures, but are considering for one formulation that contains analyte at 10ppm (yes, an active).

We do use internal standard in our GC assays of hand sanitizers, which are very high in ethyl alcohol.

[tlahren]

Hello,

I have some questions about the use of internal standards in assay determinations. If I understand correctly, we use them when the analytical method (hplc method) lacks sensitivity.

I would not say that IS will increase sensitivity. Sensitivity is purely a function of signal to noise (Peak Height of analyte over height of surrounding noise). However, using IS and surrogate standards may allow for a more accurate and precise quantitation of unknowns by correcting for extraction losses (surrogates) and reducing variability due to injection variations (IS) as well as correcting for other artifacts.

As DR stated, IS's are usually used to prevent some sort of bias in extraction, injection or other sample handling process. I generally use "surrogate IS" for standard compounds spiked into a sample prior to an extraction or other process. The surrogate is then used to observe and possible correct for sample loss (negative bias) during the process/extraction. I use the term "Internal Standard" for a compound I spike into a sample (and calibration solutions) just prior to injection to the instrument. These standards allow to correct for instrument variability issues which are usually from injection variability. Other people may use different terms for these functions so it is important to understand what you want to achieve and how to get that with use of standard compounds.

In general, my advice is to use them whenever possible or practical. I understand that they aren't always cost effective (for a good IS anyway).

[Klaus I.]

Another application for using internal standards is the compensation of injection volume variances e.g. manual injection instead of using an auto sampler.

[lmh]

They're really good for detectors that lose sensitivity over the course of time (for example, a mass spec that's gradually getting dirty as more samples are injected).

[tom jupille]

Actually, if there is a sensitivity problem, internal standardization can make things worse.

Look at it this way: imagine that you have two quantities which are subject to errors and those errors are correlated (i.e., an error in one of the quantities implies a proportional error in the other). If you take the ratio of those quantities, the errors will cancel (if the errors are perfectly correlated, the errors will cancel exactly -- unfortunately, real life is never that neat) and the percentage error in the ratio will be less than the percentage errors in the individual quantities.

On the other hand, if the errors are uncorrelated (orthogonal), when you take the ratio, the errors will accumulate and the percentage error in the ratio will be more than the percentage errors in the individual quantities (if the errors are perfectly orthogonal, then the percentage error in the ratio will be the square root of the sum of the squares of the individual percentage errors -- but again, real life is never that neat).

So what kinds of problems will affect the analyte and the IS the same way (correlated errors, remember)? Things like dilution, injection volume, extraction yield (assuming the IS and analyte have similar properties), ionization efficiency in the MS interface (assuming the IS and analyte elute close together and have similar properties). Those are situations where internal standardization can help.

What kinds of problems will affect the analyte and the IS differently (and generate uncorrelated errors)? Things like integration parameters, peak shape, excessive baseline noise (or low S/N ratio generally). Those are situations where internal standardization can make things worse.

[an_k]

Many thanks!

[lmh]

I was about to explode when I read the first line of Tom's posting, then I realised it was Tom, read the rest carefully, and of course he's utterly right.

The worst-case scenario is to pick an internal standard with diametrically opposite qualities to the analyte of interest. A while back I was asked to use an acidic compound as an internal standard for a basic analyte. It really worried me because if there is any pH-dependent effect on detection or efficiency of extraction, it's quite likely to affect the two compounds in opposite directions. This means the internal standard could magnify the problem, more so than a mere root-2 issue of orthogonal effects.

(aside: that word always bothers me. Is Orthoganol an dihydroxy-alcohol with its hydroxyl groups at 90-degrees?)

[Klaus I.]

I was about to explode when I read the first line of Tom's posting, then I realised it was Tom, read the rest carefully, and of course he's utterly right.

[tlahren]

I was about to explode when I read the first line of Tom's posting, then I realised it was Tom, read the rest carefully, and of course he's utterly right.

I had to read it twice too. Then I realized the same thing. That Tom is right. It is absolutely crucial to choose a suitable IS if one had decided to use IS for quantitation. Right now I am doing isotope dilution (13C-labeled IS) with all my analyses so the IS responds almost identically to the analyte in every way.

I always had a problem when running EPA 8270 as all the IS compounds were deuterated PAHs. They don't represent the chemical behavior or chromatography of most of the compounds in the full SVOC list.

[Klaus I.]

I was about to explode when I read the first line of Tom's posting, then I realised it was Tom, read the rest carefully, and of course he's utterly right.

I had to read it twice too. Then I realized the same thing. That Tom is right. It is absolutely crucial to choose a suitable IS if one had decided to use IS for quantitation. Right now I am doing isotope dilution (13C-labeled IS) with all my analyses so the IS responds almost identically to the analyte in every way.

I always had a problem when running EPA 8270 as all the IS compounds were deuterated PAHs. They don't represent the chemical behavior or chromatography of most of the compounds in the full SVOC list.

This questions is real funny, nobody have activeley tried to understand the problem of the original poster. But everbody have an Answer for the Posting. Only Tom mades a abstract for commun applications. But then other peoples become a little bit angry...

Nevertheless, we don't know anything about the requested applications.

[danko]

In my opinion, the standard addition technique is often a better alternative to the IS use, in many situation when IS is relevant.

Best Regards

[an_k]

Thank you all for important information about the correct choice of an i.s and whether it has to be used or not.
But my question is set in a much lower basis:

Does the general principle % assay = (Cstd x Asample) / (Csample x Astd) x 100 applies in such a case?

I work on the evaluation of such a method developed by an external lab. Lets say that internal standard was chosen correctly (enen though it is not really justified quite well).

But in the point where the assay formula has to be excluded, can we use the above known type for assays, when ''Area'' is in fact ''the ratio of areas'' of analyte signal and internal std signal?
I' m trying to avoid as much questions by the authorities as possible, and also to understand the analytical quality of the method i review.
The laboratory developed and validated the method but it is not possible for them to exclude a calculation formula!

So i'm trying it for them and since i'm not experienced in working with internal standards, i' m asking..