Internal standard quantitation

General information

Internal standard quantitation uses peak tables prepared from the standard, similar to the External standard quantitation. However, a fixed quantity of an additional component is added to every separation run, including the sample. The peak sizes of the standards and the sample are then related to the peak size of the internal standards to compensate for any changes that may have occurred between the runs.

General assumption	The internal standard technique relies on the assumption that any changes in the injected amount of the component(s) of interest, e.g. due to sample preparation losses, correspond to equal changes in the injected amount of the internal standard component.

Advantages	Internal standard quantitation reduces errors that are caused by changes in the system between successive runs with the sample and the standard concentration levels. For example, there may be unpredictable losses during the sample preparation procedure or unintentional changes in the amounts that are injected.

What is a suitable internal standard?

A suitable internal standard must meet the following conditions:

It must be well separated from the components in the sample (not just from the components of interest).
It must not be present naturally in the sample(s).
It must have similar chemical properties to the component(s) of interest.

To be able to compensate for losses during the sample preparation, all the standard concentration levels must be subjected to the same sample preparation procedure as the samples.

Note: If there are several components of interest, they must all be chemically similar.

How to perform Internal standard quantitation

The table below describes briefly how Internal standard quantitation is performed.

Step	Action
1	Prepare a series of concentration levels from the standard.
2	Add an additional component, the internal standard, in the same concentration to all the standards and to the sample.
3	Perform a run for each standard and the sample.
4	Integrate the curves to produce a peak table for all standard runs and for the sample. Result: Each curve contains a peak from the internal standard. Changes in the size of the internal standard peak indicate changes in the system. See illustration below:
5	Plot all peak sizes relative to the size of the internal standard peak to produce a calibration curve for each component. The standard peak area, relative to the internal standard peak area, is used to produce a point on the calibration curve. See illustration below:
6	Prepare data from the sample in the same way as the data from the standard runs to produce peak sizes relative to the internal standard peak size. The resulting relative value is applied to the calibration curve to determine the amount and concentration of the component of interest. See illustration below:

Reliability

Internal standard quantitation is potentially the most reliable of the quantitation techniques. However, if the internal standard component is not selected carefully, the reliability will probably be worse than with the external standard technique. There are some specific factors that can affect the reliability:

There is an increased risk of overlap when the extra component (the internal standard) is added if the sample contains many peaks.
The addition of the internal standard must be accurate in both the standards and samples, otherwise, the precision of the quantitation will be reduced dramatically.

2005-06-15