Chromatography Forum

Is it always necessary to use the internal standard in HPLC Quantitative analysis?

Is it always necessary to use the internal standard in HPLC Quantitative analysis?

No

Is it always necessary to use the internal standard in HPLC Quantitative analysis?

No.

We NEVER used internal standard for our HPLC analyses, and I developed and cGMP-validated many assays using external standard technique for our OTC products; of course autosamplers were used.

For GC, we probably used internal standard maybe 5% of the time.

It depends on the sample preparation. If it is just a dilute and shoot you do not need an internal standard. If it involves derivatization, Liquid liquid extractions, drying and redissolving, SPME for sure I would definitely use an ITSD. For stuff like Quechers some of the extractions are 60% recovery so they have to use an isotopically labeled ITSD because you can get away with analytical murder with those.

To expand a bit on the previous replies:
If the precision is adequate when using external standardization, then an internal standard is unnecessary, and may even make things worse. If the precision is not adequate, you have to make a judgement regarding the largest contribution(s) to error.
If those are in the "upstream" part of the workflow (e.g., sample preparation, injection, etc.) then internal standardization can be an improvement (the internal standard will tend to cancel errors).
If the dominant sources of error are in the "downstream" part of the workflow (e.g., integration errors, resolution issues, noisy baseline, etc) then internal standardization will probably make things worse (internal standard errors will add to the total error).

In the environmental monitoring field we use two different approaches. We add a "surrogate" to the unextracted sample which is a compound similar to the target analyte to monitor extraction efficiency, and then add an internal standard to the final extract for quantitation and to correct any problems with injection differences or final matrix effects and such.

If the internal standard recovers well but the surrogate is low, then you know there is a problem in the extraction/preparation step.

If the internal standard is high/low then you know there is a problem with the injection or enhancement/suppression from the matrix or other such problem but the internal standard can compensate and result in more accurate quantitation so there are fewer reanalysis required.