Key Considerations for Absolute Quantitation

Posted on September 27, 2019


Internal Standard Curves

The sensitivity of an experiment is typically described by the limit of quantitation (LOQ or LLOQ). In the absence of matrix, this value is related to the overall sensitivity of the analytical instrument (generally a mass spectrometer). However, when matrix and internal standards are present in the sample, the sensitivity of the assay can be significantly affected in a way that is independent of the instrument configuration. Interference from isobars present in the matrix can increase the LOQ simply by raising the background. This can sometimes be mitigated by changing chromatographic strategies, but generally speaking, the matrix effects can be a significant challenge to overcome and will increase the LOQ. This aspect of quantitation is well understood and typically characterized during method validation, and its effects on the LOQ—as well as the accuracy—of the assay are determined. However, an issue that typically fails to garner as much consideration is the effects that internal and primary standard purities may have on the LOQ, accuracy and the dynamic range of an assay.



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An internal standard curve is typically established by spiking a known amount of internal standard to increasing amounts of a primary reference standard in the presence or absence of matrix. A plot is generated that relates the ratio of the peak areas (analyte/IS) to the ratio of their respective concentrations ([analyte]/[IS]). The resulting slope can be used to calculate the concentration of the analyte in samples. The example shown here is an ideal case with a linear instrument response of ~5 orders of magnitude. The R value squared is a goodness-of-fit measure for linear regression models

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A more realistic internal standard curve is modelled here. In this case, the upper and lower concentrations of the primary reference standard are expanded to showcase the upper and lower limits of quantitation. Accurate quantitation can only be determined from the linear portion of the curve. The sigmoidal nature of the curve shows the upper limit of quantitation, which is typically due to saturation of the instrument detector. The LLOQ, as mentioned above, reflects limits in the sensitivity of the instrument and potential matrix effects. Care must be taken to ensure the assay is designed so that the concentration of the target analyte falls within these limits.

Effects of Standard Purity on Standard Curves

Matrix and instrument effects aside, there are several issues that must be considered regarding the primary reference and internal standards used for absolute quantitation during method validation. The first consideration is the design of the labeled internal standard. The incorporated stable isotope must move the mass of the IS away from the mass of the analyte in such a way that, 1) the isotopes of the analyte do not overlap with the IS, and 2) the isotopic purity of the internal standard is sufficient such that there is no incompletely labeled IS. In regards to the first statement, the mass of the IS must be shifted far enough away from the analyte to prevent its isotopes from overlapping with the IS. This requisite mass difference depends on the types of elements in the analyte, the numbers of carbons and sulfurs, degree of unsaturation, and double bond variants. This is especially important if the label—D or 13C—is not incorporated in the identifying Q3 fragment. Ideally, the IS should be designed such that both precursor and product ions are shifted away from the analyte, especially when using less sophisticated instrumentation. Finally, the issue of isotope scrambling during fragmentation can confound results and ultimately affect quantitation.

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When the MAnalyte = MIS, which can occur when the IS has poor isotopic purity, the LLOQ increases. As shown in the figure below, increasing the LLOQ not only affects the sensitivity of the assay, but it also reduces the dynamic range. Isotopic purity refers to the amount of internal standard that is fully labeled with the stable elemental isotope. For example, an IS that is designed with 3 deuterium atoms meets the first requirement that the mass be sufficiently shifted away from the analyte mass, but if its isotopic distribution is 76% D3, 12% D2, 7% D1 and 5% D0, the mass of the IS with D0 equals that of the analyte and thus increases the LLOQ. At Avanti, we typically label compounds with ≥ 5 deuterium to avoid this issue. We also make every effort to put deuterium labels in locations that are not subject to H-D exchange, such as carbons β to carbonyls. As a consequence, our labeled standards typically have > 90% isotopic purity. Even if isobaric interference between the IS and the analyte is not an issue, a low isotopic impurity ultimately lowers the ULOQ, which again affects the dynamic range of the assay. The second and most critical consideration for any internal strategy is to use the highest purity primary reference standards available. The accuracy of the assay is completely dependent on the purity and hence concentration of the primary reference standard. At Avanti, we make and sell standards that are > 99% pure—the Good Stuff. When using our standards in a quantitative assay, you can be confident that their purity will not be a limiting factor in your analyses. Every compound we sell has an experimentally-derived purity — be it structural or isotopic — that is stated in our catalog.