INTERNAL STANDARD AND EXTERNAL STANDARD
DISCRIPTIONAn internal standard in analytical chemistry is a chemical substance that is added in a constant amount to samples, the blank and calibration standards in a chemical analysis. This substance can be used for calibration by plotting the ratio of the analyte signal to the internal standard signal as a function of the analyte standard concentration. This is done to correct analyte losses during sample preparation. The internal standard is a compound that must be show similar behaviour to the analyte.
This ratio for the samples is then used to obtain their analyte concentrations from a calibration curve. The internal standard used needs to provide a signal that is similar to the analyte signal in most ways but sufficiently different so that the two signals are readily distinguishable by the instrument.
An external standard is like the internal standard (known behaviour), but is not added to the unknown. Rather it is run alone, as a sample, and usually at different concentrations, so you can generate a standard curve. Again, the peak areas are related to the known amounts of external standard run. External standards do not correct for losses that may occur during preparation of the sample, such as extraction, centrifugation, evaporation, etc. Internal standards would correct for this if added to the unknown at the beginning of the sample preparation.
Internal standard methods are used to improve the precision and accuracy of results where volume errors are difficult to predict and control. A systematic approach has been used to compare internal and external standard methods in high performance liquid chromatography (HPLC). The precision was determined at several different injection volumes for HPLC and ultrahigh-pressure liquid chromatography (UHPLC), with two analyte and internal standard combinations. Precision using three methods of adding the internal standard to the analyte before final dilution was examined. The internal standard method outperformed external standard methods in all instances
In an external standard calibration method, the absolute analyte response is plotted against the analyte concentration to create the calibration curve. An external standard method will not provide acceptable results when considerable volume errors are expected because of sample preparation or injection-to-injection variation. An IS method, which is a method where a carefully chosen compound different from the analyte of interest is added uniformly to every standard and sample, gives improved precision results in quantitative chromatographic experiments. The internal standard calibration curves plot the ratio of the analyte response to the internal standard response (response factor) against the ratio of the analyte amount to the internal standard amount. The resultant calibration curve is applied to the ratio of the response of the analyte to the response of the internal standard in the samples and the amount of analyte present is determined.
Several approaches have been used to determine the amount of internal standard that should be used in preparing the standards and the samples, but none have illustrated definitive results (1–4). For example, Haefelfinger (1) reports that the IS peak height or area must be similar to that of the analyte of interest, but does not present supporting data. Araujo and colleagues (2) show that experimental design strategies can be used to determine the optimal amount of internal standard used while Altria and Fabre (3) show that the IS should be used in the highest possible concentration.
Calculation of the response factor assumes that the detector gives a linear response for both the analyte and the internal standard over the entire range of the experiment. Since this is not always the case, it is essential to understand the behavior of the response factor as the concentration or amount of analyte and internal standard are varied. Knowing the behavior of the response factor allows one to set limits on the useful range of the chosen analyte or internal standard concentration combinations.
The internal standard method is used to improve the precision and accuracy of results where volume errors are difficult to predict and control. Examples of types of errors that are minimized by the use of an internal standard are those caused by evaporation of solvents, injection errors, and complex sample preparation involving transfers, extractions, and dilutions. An internal standard must be chosen properly and a known amount added carefully to both sample and standard solutions to minimize error and be utilized to its full advantage. The resulting internal standard peak should be well resolved from other components in the sample and properly integrated. If all of these conditions are not met, the use of an internal standard may actually increase the variability of the results. One report suggests that whenever detector noise or integration errors are the dominant sources of error, the use of an internal standard will likely make the results of the experiment worse (5).
A paper published by P. Haefelfinger in the Journal of Chromatography in 1981 (1) discussed some limitations of the internal standard technique in HPLC. Using the law of propagation of errors, the paper showed conditions that need to be met for the internal standard procedure to improve results. In addition to the mathematical illustration, Haefelfinger detailed practical examples where either internal or external standard methods were advantageous.
The Journal of the Pharmaceutical Society of Japan published a study in 2003 (6) that found that the internal standard method did not offer an improvement in precision with the then current autosampler technology. Interestingly, they also found that if the peak of the internal standard was small, the relative standard deviation (RSD) was actually larger than the RSD for the external standard method (6). The limitation of this study was that only one injection volume (10 µL) was used to establish the conclusions.
In our work, a systematic approach has been used to compare the internal to the external standard method using two analytes and two internal standards. The precision resulting from both an internal and external standard method were determined at several injection volumes and on two different instruments. Three methods of adding the IS to the analyte before final dilution have been compared. In the first, a solid internal standard was weighed directly into the glassware containing the sample before dilution with solvent. In the second, a solution of a known concentration of the IS was prepared and a known volume of this solution was added to the sample prior to dilution. In the third, the IS was added in the same manner as the second method, but the internal standard solution was weighed and the weight, not the volume, was used in the IS calculations. We examined the effect of weight of analyte and internal standard on the precision of the results. Initially, the weights of the analyte were varied versus a constant IS concentration, and then the concentration of the internal standard was varied versus a constant weight of the analyte.
Standard deviation was chosen to monitor precision. All possible errors are reflected in the standard deviations of the final measurements, including each step in the sample preparation, sample transfer, and sample introduction into the HPLC or UHPLC system, as well as the HPLC or UHPLC analyses themselves. Both external and internal standard calibration methods were used to calculate the percent recoveries for comparison.
Choosing an Internal Standard
When creating a new method for quantitation, the choice of the correct internal standard (IS) can improve the accuracy and precision of the method. The proper internal standard should be chemically similar to the compound(s) that you are analyzing, but is not expected to be naturally present in your sample. It is best to choose compounds that have the same functional groups, boiling points, and activity as your target compounds.
Two examples
If you are using MS, then it is common to use a deuterated analog of your compound of interest i.e. Amphetamine & Amphetamine d-5.
If you are working with a non-MS detector, the deuterated internal standards would coelute with your analyte of interest and cause problems with quantitation. In this case, you would try to use a compound that is somewhat chemically similar, but would not be found in your sample, i.e. if your target is tri-chlorophenol, you might use a tri-bromophenol or a di-chlorophenol internal standard.
Any time you choose in internal standard, you have to validate that it does correct for small amounts of variation within the analytical process. The best quantitation is achieved when with an internal standard for each one of your target compounds, but in many cases this is impractical because of the number of analytes in your mix and/or the cost of each internal standard.
Important Fact -
Adding an internal standard (IS) to an assay can be an excellent way to often improve method precision and accuracy. An IS can account for signal suppression (or enhancement), that may be caused by the sample matrix. When using an IS, the response of your target compound(s) is compared to the response of the IS. The internal standard is added at a constant amount to all standards, controls, and samples. Since it is a constant, the IS can be monitored to determine if an individual injection is valid. Additionally, the IS can be used to calculate relative retention time and assist with peak identification.
Choosing a proper internal standard is the key to the success of using one. The IS should be chemically similar to your target compounds, and it should not be present in your sample(s). I would recommend reviewing your target compound’s structure and boiling point and choose an IS that would be similar. Ideally, one would have an IS for each target compound in one’s method, but this can become impractical with large compound lists. In these cases, choose a handful of compounds to match early eluting, mid-eluting, and late eluting compounds in your assay.
If you are using a mass spec detector, an isotopically labeled analog of your target compound makes an excellent IS. These are compounds that are deuterium labeled (contain deuterium in place of hydrogen) or 13C labeled (contain 13C atom instead of 12C atom). An example would be using Atrazine-d5 as an IS for Atrazine or other triazine herbicide compounds. Isotopically labeled analogs will chromatograph extremely similar to their non-labelled counterparts.
The drawback to isotopically labeled internal standards is that they are not available for every compound. Additionally, they coelute with the non-labelled target compound; hence, they can only be used with mass spectrometry. With a GC detector, such as an FID, ECD, or NPD or LC detector, such as UV or Fluorescence, these coelutions will lead to headaches in the lab. In these cases, one needs to look at for an internal standard that is similar to the target compound but not found in your sample. Many environmental assays use an internal standard that contains bromine or fluorine when analyzing chlorine containing compounds. As an example, a compound such as 4-bromofluorobenzene could be a suggested IS for chlorobenzene analysis. In the case of medical cannabis potency testing, my colleague Jack Cochran used compounds somewhat similar in structure and behavior to the cannabinoids that were present.
