Analyte and internal standard cross signal contributions and their impact on quantitation in LC–MS based bioanalysis

Calibration graphs in isotope dilution mass spectrometry

“…However, despite the fact that the above least squares approach for Padé [1,1] does not yield the lowest residual regression variance possible, it nevertheless outperforms polynomial functions (see Figure 2). Consequently, the approximate nature of linear fitting with Padé[1,1] does not impede its usefulness.…”

“…It is important to reiterate that, unlike the polynomial equations which are empirical approximations of the isotope dilution curve, Padé [1,1] function provides an exact description of the curve. Consequently, the use of Padé equation for isotope dilution will not incur additional errors due to the arbitrary choice of the model equation.…”

“…In addition, one does not have to resort to a larger number of fitting parameters in order to describe more pronounced curvatures as it is practiced with polynomials. Last, but not least, Padé [1,1] is a monotonous function and does not have inflection points in contrast to polynomials. All of these properties make Padé [1,1] reliable not only for interpolation but also for extrapolation which is useful in the method of standard addition [12].…”

“…Frequently, in order to calculate the amount of the analyte in the sample, a graphical approach is employed whereby the resulting isotope ratios are plotted against the mixtures of the isotopic standard with known amounts of natural standard (Figure 1). In situations when the resulting curve is not sufficiently linear due to the significant spectral overlap between the analyte and isotopic standard [1], polynomial empirical functions have been proposed to approximate the curvature of the isotope dilution function.…”

“…Albeit half-percent may indeed be an acceptable error to many, analysts should be aware that there are better functions available for isotope-based calibration when dealing with nonlinear calibration plots. This note brings attention to the fact that the use of polynomial curves in isotope dilution introduces unnecessary errors which can be avoided by using rational Padé [1,1] equation and linear least squares fitting.…”