Rationale: Regioisomeric analysis of triacylglycerols is important in understanding lipid biochemistry and the involvement of lipids in disease and nutrition. The use of calibration plots employing fractional abundances provides a simple and rapid method for such analyses. These plots are believed to be linear, but evidence exists for non-linearity. The behavior of such plots needs to be understood to allow for proper interpretation of regioisomeric data., Methods: Solutions of five regioisomer pairs were prepared from pure standards and used to construct calibration plots using triple-stage tandem mass spectrometry (MS(3) ) with electrospray ionization (ESIMS(3) ) and cationization by lithium ions. The data were taken by direct infusion with an AB SCIEX QTRAP 2000 QqLIT mass spectrometer., Results: Non-linear calibration plots were observed for the four isomer pairs containing the polyunsaturated eicosapentaenoic (20:5) and docosahexaenoic (22:6) acids paired with palmitic acid (16:0) or myristic acid (14:0), while the pair including palmitic and stearic (18:0) acids provided a linear plot. A non-linear model was developed for these plots and then verified experimentally., Conclusions: The fractional abundance calibration plots used in regioisomeric analysis of triacylglycerols are intrinsically non-linear, but may appear linear if the scatter in data points obscures the curvature, if the curvature is slight, or if the response factors for the two isomers in the regioisomer pair are similar. Therefore, linearity should not be assumed for these types of measurements until confirmed experimentally., (Copyright © 2013 John Wiley & Sons, Ltd.)