Comprehensive investigation of matrix effect evaluation approaches for reliable quantification in bioanalysis using UHPLC-MS/MS
- Anal Chim Acta. 2026 Sep 1:1413:345642. doi: 10.1016/j.aca.2026.345642.
- 1. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Heyrovského 1203, Hradec Králové, 500 03, Czech Republic.
- 2. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Heyrovského 1203, Hradec Králové, 500 03, Czech Republic. Electronic address: [email protected].
Background: 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.
Results: 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/X0, 1/X, and 1/X2 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.
Significance: 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.