Your question is not very clear. To calculate internal standard you have to first create a diagram of relation between your substrate and internal standard, then use it and the concentration of the solution (your sample) to calculate the amount of internal standard as well as your substrate in your sample. 

I am not shore what you mean. In general we add internal standards to analyte solutions in order to compare results more accurately, for this you can divide the area of the analyte by the area of the internal standard of the same analysis and use this value instead of the analyte area. I suggest to dissolve all samples and standards in a solution of the internal standard to reduce variation by the internal standard addition.

it depends on what you want to calculate exactly. If you use the internal standards for quantifying, you do an internal calibration and add the same concentration of internal standard, as a reference, in all your injection vials (calibration and samples). After, you use the ratio between area of IS / area of compound to do the calibration curves to quantify your samples. If you want to use a standard solution to evaluate an extraction recovery, it is not an internal standard but a surrogate standard added to the sample at a known concentration in order to determine extraction efficiency. To do that, you make a calibration and quantify this surrogate as your target compounds.

I agree with Catherine. If what you want is to assess percentage recovery, an external calibration is necessary.

And to evaluate matrix effect, you must use internal standard

If you want to assess recovery from a certain matrix for a compound that you then intend to use as an internal standard for other samples in the same matrix, you need to add a known amount of your standard to your matrix and apply the sample prep and analytical method that you intend to apply later to all samples. You then calculate your recovered amount using a calibration curve obtained by injecting solvent solutions of your standard directly into the analytical instrument (no sample prep).  That way you determine the recovery of your sample prep for that particular compound from that particular matrix. You will need a number of repetitions and I would do this for at least 2 concentrations of the standard in the matrix, as close as possible to the concentration you intend to use as an internal standard. Keep in mind that this recovery is only usefull if all the samples for which you intend to use this compound as an internal standard are in the exact same matrix.

With the desire to measure recovery of the internal standard, I presume that you are adding the internal standard at the start of sample preparation.  (Some spike at the end and call it an internal standard.)  The trick is to measure it relative to a compound which can not change during the extraction process.  This would be a compound added into the GC vial just before derivitization (if used) and instrumental analysis.  We can call this the recovery standard.  So, spike several samples with internal standard and prepare them.  Also prepare several samples with no internal standard included.  At the end of preparation add an aliquot of recovery standard to all and internal standard to the extracts of samples which did not have internal standard added at the start.  And have a few samples which never got inernal standard added at all.  This will account for matrix effects in the analysis (as opposed to using solution standard mixtures vs. an extract) and will provide a measure of any peaks that would interfere with the measurements.  This approach can be used for calculating recovery of analytes as well.  We can ratio the internal standard to the recovery standard where the internal standard was added at the start or at the end.  The ratio of internal standard response in a sample with only recovery standard added is important as it gives a measure of response that is not from the added internal standard, but is part of the measured responses.

thank you all for responding me............

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