Minimal Number of Internal Standards for Quantification

In Section 13.3.2, the importance of the mass levels of internal standards used for quantification is extensively discussed. In fact, the number of internal standards used for quantification of lipid species of a class is equally critical. In this section, a minimal number of internal standards that should be used for quantification of the species of a class by different approaches are discussed. In summary, the number of internal standards that should be used really depends on the number of the variables present in the employed approach. 

In the SIM method after LC-MS with isocratic elution, the additional variable in comparison to that conducted in the full mass scan mode after direct infusion is the changes in the concentration of individual species. Therefore, two or more internal standards at different elution times are necessary in this case. If a gradient of mobile phase is used for elution, an extra variable is introduced and at least one additional internal standard should be employed. In the case of an SRM/MRM method after 

TABLE 15.1 Summary of Variables Present in the Methods and Their Required Minimal Number of Internal Standards for Quantification of Individual Species of a Polar Lipid Class 

Platforms Variables Minimal Number of Internal Standards 

one additional variable exists in comparison to that of the SIM method after LC-MS, that is, the tandem MS process. Therefore, an extra internal standard should be employed relative to the SIM method. In both SIM and SRM/MRM methods after LC-MS, the necessary number of internal standards employed for accurate quantification could be compensated with a combination of an internal standard with a few external calibration curves. 

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Taken together, the minimal number of internal standards that should be used for accurate quantification is varied from method to method, largely depending on the existence of the variables in each method. Table 15.1 summarizes the easily recognizable variables present in each method and therefore the minimal number of internal standards necessary for the methods. As aforementioned, some alternative approaches could be used to reduce the minimal number of internal standards for quantification. It should be pointed out that any method employing a number of internal standards much less than the variables present in the method is unlikely able to provide accurate quantification, but it can still be used for relative comparison between the samples. If this is the case, it is advisable that the investigators should not overstate the results as quantitative. 

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