The internal standard should be added as soon as possible in the analytical protocol.  So before SPE.

The internal standard  should be added before the solid phase extraction step to allow similar treatment as the analyte.

If you add the IS after SPE you are only going to correct for instrumental fluctuations. Consequently, it is always better to add it from the very beggining of the analytical procedure. In this case, it is known as a "surrogate".

The only drawack about adding it prior to sample analysis is  that you'll need a higher ammount of IS. 

I hope that this helps you. 

Neus correctly identifies standards that are put through the extraction process as "surrogates" which are often used to quality control the extraction process. It often provides useful information on the performance of an analytical method to include both surrogates and an internal standard  that is not put through the extraction process. In particular this gives good information on trends in the extraction efficiency which are difficult to follow unless an extraction-independent calibration is also available. If things go wrong this helps isolate the source of any problem as being an instrumental one or associated with the extraction process. 

As indicated by the above discussion the nomenclature around 'internal standards' is not clear.  Different people call these compounds different things based on when they are added in the analytical process.  "Internal Standards" can be added as late as immediately before injection - in which case they are sometimes called "injection standards".  In general if your internal stds are going to be used to correct for extraction and clean-up recoveries then they should be added as early in the process as possible.  If the internal Standard is being used only to provide a reference for instrumental detection variability they can be added immediately before injection.  In some analyses (e.g. dioxin) multiple "internal standards" or standard mixtures are added at various points in the analytical process to account for all possible sources of variability.

I recommend that you quantify the recovery of your internal standard (or candidate internal standards) and analyte(s) to confirm that they are operating as a suitable internal standard by mimicking the analyte in the SPE process and beyond.  If you screen the wash solutions you may detect whether your washing is removing what you want to keep.

Thank you for your answers. Basically, in our experiment, we tried using Terbutylazine-2-hydroxy as an internal standard for pharmaceuticals that we want to analyze. We are actually trying to quantify concentrations of different pharmaceuticals in river water. Now, we are in the process of doing a calibration curve which will have concentration vs ratio (of the areas of our analytes/ area of our internal standard). We already added the IS before SPE but it gave different area readings in LC-MS/MS (QQQ) which in turn did not give good results in the ratio (thus, low r^2). I am just wondering if it should be added right after SPE to normalize our values. Please enlighten me some more. Thanks!

I suggest a step wise approach. First  check the reproducibility  of the determination of your target  compounds in your final SPE eluant at around the concentration expected in your  final injection sample. This will tell you if your instrumental method is OK. Then put the same compounds in “pure water” through the SPE add your internal standard afterwards. Do several runs and check the reproducibility of your extractions, you could usefully add candidate surrogates  along with the analytes. Then do the same but spike your analytes into river water, ideally  a sample with  undetectable analyte concentrations. Again do this repeatedly and  check the reproducibility. Then take a good hard look at the data and you may be able to see where things are going wrong. You could also check the river water samples to see if there is any overlap with analytes or internal standard  from interferents in the river water that is not being excluded by the selectivity of your MS detector (unlikely but worth a look). Finally are you sure that you are not just working too close to the detection limit? As the LOD is approached the relative precision degrades significantly and this could really throw out your calibration curves to the extent that you cannot even get a reasonable fit.

terbutylazine is a pesticide used worldwide, it is as unlabelled compound not among the best possible choices.

Generally internal standards can be used to controll procedure (procedural IS) and should be then added at the earleist possible stade or as calibration IS (possibly just added before injection).

It is obvious that the procedural IS controlls considerably more possible errors and uncertainties than the calibration IS

if you are using HPLC-MS/MS you should work with isotope labelled internal standards so you can perform isotope dilution measurements, which will increase precision and correctness considerably.

If your r2 in your calibration is not good something is wrong either with the instrument or the calibration solutions.

After SPE

Once again thank you for your answers. Meanwhile, our group has already come up with a calibration curve (before SPE) with a range of 5ppb-1000ppb. All equations for standards have been developed already and r^2 of the analytes are greater than .99. Also, predictability of the analyte concentration is very near with that of the expected concentration. Everything seems okay but the calibration curve after SPE. We are using LC-MS/MS (Agilent QQQ) for our analysis. are internal standards/labelled compounds really necessary?

Spiking / recovery exp or addn of internal std must be after SPE. AND should be compared with calibration after SPE.

My brief answer to the question is, yes, internal standards should be added before the SPE extraction; i.e., the internal standards should undergo the same sample preparation procedure as analytes being assayed.

Depends on your choice of absorbents in the SPE catridges/centrifuge tubes. If working with PSA (Primary Secondary Amines), Internal standard should be added after SPE. There is likeliness of absorption of internal standard (especially the TPP) by PSA which would affect the quantifications. This is especially applicable where measurements on Internal Standard are involved in final calculations.