Internal Standard (ISTD) HPLC Calculation Notes:

Why Use an ISTD?

Two main reasons: (1) The addition of an internal standard to all vials containing standards and any unknown samples takes into account any changes caused by the sample preparation process. This is useful when the samples are run through various media or filters as part of a pre-treatment or clean-up initial phase (e.g. clinical or biological samples). (2) Retention time drift over the day can be compensated for through the use of ISTDs.

Key Points To Keep In Mind: 

• The ISTD must be of known purity (by certified method), have a similar response as the sample and not interfere with the analysis.

•  The calculated amount of your unknown samples is directly related to the amount of the ISTD used. The results are calculated based on the ratio of the responses for both peaks (i.e. std and unknown). The amount of ISTD used in all vials must be kept constant.

• Relating peak retention times based on the ISTD (as a ratio) instead of establishing retention time windows makes it much easier to transfer methods to other systems and also account for variation seen. This is because with ISTD and Relative Response Ratios you can define peak retention based on the elution time of the ISTD and not the actual retention times. 

• You must use identical integration parameters to calculate the areas of the standards, samples and/or unknowns. Failure to do this may invalidate the process.

• For Multi-level Calibrations you will calculate an amount and response ratio. You will do this for each calibration level (and each std type if multiple standards are used). Note: Most professional chromatography data systems are designed with special fields for the internal standard data and will perform these calculations for you once you load the data for each level into the calibration table. Response ratios are then used for measurements (Response Ratio = Sample/Std Area / ISTD Area). The accuracy of your calibration curve fit and the overall reproducibility of the entire method used will impact your final results. Poor quality curve fit and/or RSD equals poor accuracy.

• Highest accuracy is achieved using a professionally developed method which first retains (with proper K prime values), then elutes all samples off the column during the run. Column wash and equilibration steps should be separate runs, not part of the analysis method. Poor quality method development is the most common reason why calibration results are poor or RSD is high. 

• Use the same injection volume for all samples (unknowns) and standards (knowns).

• Base the amount of ISTD concentration such that it is between 1/3 and 1/2 of the expected concentration of the sample(s). *The sample's target range is best.

Calculations:

Response Ratio = Sample Response (Area) / ISTD Response (Area)

Response Factor = Amount Ratio / Response Ratio

Summary:

The best way to guarantee success and generate high quality data is to first develop a stable and chromatographically correct HPLC method which resolves apart all of the compounds AND elutes everything off the column during the run. Do not start the calibration or quantification process until the method has been demonstrated to be stable, reliable and working perfectly. Problems seen with calibration methods are often caused by poor quality methods. Invest the time needed to produce an excellent quality method first and then you should have few to no problems later on.

*Related Reading: "External vs. Internal Standard Calibration in HPLC";