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Matrix effect (ME) evaluation is an indispensable part of the method development and validation when using ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS). The rotational and translational matrix effects are often observed. The rotational matrix effect depends on the analyte concentration and affects the slope of the calibration function, while the translational matrix effect is independent of the analyte concentration and affects the intercept of the calibration function. Two matrix effect evaluation strategies, the post-extraction addition and the comparison of calibration curve slopes, are the most widely used for the quantitative ME evaluation. The post-extraction addition approach recommended by the European Medicines Agency (EMA) guideline is considered the reference approach. However, the extent to which these approaches provide comparable results has not been systematically assessed. We evaluated the suitability of the calibration curve slope approach for quantifying ME. Two ME evaluation strategies were systematically compared using ESI-UHPLC-MS/MS in both negative and positive ionization modes for the analysis of 26 compounds in serum. Various calibration models, including 1/X 0 , 1/X, and 1/X 2 weighting or logarithmic transformation, were assessed. None of the tested models provided overestimated results compared to the post-extraction addition approach. However, several models exhibited underestimated results. Thus, we developed a new approach for calculation of ME taking into account also the translational ME expressed by the intercept of the calibration curve. The accuracy of the new approach was subsequently determined using three different matrices and two different instrumental platforms. It is not advisable to rely solely on the calibration curve slope approach for accurate estimation of ME unless translational ME derived from calibration curve intercept are also involved in calculation. Therefore, a novel intercept-based equation was proposed for the calculation of translational matrix effects. The total matrix effect, obtained as the sum of the slope- and intercept-derived contributions, closely corresponds to matrix effects determined by the post-extraction addition approach. • Two matrix effect quantitative evaluation strategies were evaluated, the post-extraction addition and the calibration curve slope-based approach. • The selection of appropriate calibration model is necessary for correct evaluation of ME. • Different ME were observed depending on the transformation or weighting model of the calibration curve slope approach. • The calibration curve slope approach resulted in underestimation of ME because it did not take into account translational ME. • A new equation allowing calculation of translational ME based on the comparison of intercepts from standard and matrix calibration models was proposed.
Vlčková et al. (Sat,) studied this question.