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Affectors of Laboratory Results: Part VI
Just a reminder, the purpose of this series is to enable one to better evaluate laboratory results in a more informed manner, whether the results are published in the media or received on samples a pharmacy has submitted for analysis. If the complete analytical method and workup is not available for scrutiny, it is difficult to be confident of the results. It should be noted that laboratories are not perfect and function the same as other businesses and professions. They, too, must be checked and held to a high level of performance. It doesn’t matter if the laboratory is in a university, independent contract laboratory, a PHARMA company, or a part of the government, they must meet high standards that are available for evaluation.
This week, let’s turn our attention to three common methods of quantitation; the External Standard, Internal Standard and the Standard Addition methods of analysis. Each has its own place in an analytical laboratory. External standard methods are used with all techniques while the internal standard methods are employed predominately with chromatographic analysis.
The external standard method is basically what it says. The standard curve is established as discussed in Part V. First, a series of standard solutions are prepared very accurately from reference standards. This series consists of different concentrations of the analyte in an appropriate solvent, i.e., 1, 2, 3, 4, 5 mg/mL. The individual standards are subjected to analysis and a response factor (absorbance, peak height, peak area, etc.) is obtained. The concentration (x-axis) is plotted against the response (y-axis) and the standard curve results. Second, the unknown sample is subjected to analysis and the response obtained and compared with the standard curve to determine its concentration. In some cases, the sample must be diluted or concentrated to come within the standard curve range and a multiplication factor must be considered in arriving at the final concentration. As is evident, the standard curve and the sample are separate in this system.
In the internal standard method, a fixed quantity of a pure substance is added to samples and standards alike. The response of the analyte and internal standard, each corrected for any background, are determined and the ratio of the two responses is calculated. If the parameters affecting the measured responses are well controlled, the response of the internal standard will not change, since the concentration of the internal standard is fixed. The response ratio now depends on the analyte concentration. The plot of the response ratio as a function of analyte concentration yields a calibration or standard curve. The internal standard method is very precise method for quantitation as it corrects for some procedure and apparatus errors. The two most common sources of errors, as we have generally discussed, are (1) variation in sample size, and (2) errors created during the sample preparation operations. It is vitally important that the internal standard be selected carefully so it will mimic the physicochemical and analytical properties of the analyte, i.e., it should be of a similar chemical structure. As is evident, since both the internal standard and the sample analyte are treated identically, there is generally greater precision in this method.
The standard addition method is performed by adding a small amount of standard solution to a portion of a previously analyzed sample and repeating the analysis using the same reagents, instrument and technique. The amount of increase in the test result should exactly equal the amount of standard added to the sample. The advantage of the standard additions over the normal standardization methods is that the performance of a specific procedure can be checked under actual operating conditions and used to detect flaws and biases in a particular method. It is especially useful when it is impossible to suppress interferences from matrix (sample) elements. The instrument response must be a linear function of analyte concentration. Standard addition is commonly used in analyses of electrolytes and trace metal elements, especially during clinical assays and pharmacokinetic studies.
In summary, for chromatographic methods, the internal standard method is preferred while in spectroscopic methods and others, the external standard method is widely used.
Next week in Part VII, we will look at individual sources of errors in a laboratory analysis and their effect (increase or decrease in analyte concentration) on analysis.
Loyd V. Allen, Jr., Ph.D., R.Ph
Editor-in-Chief
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