The importance of complete overlapping of analyte and internal standard peaks in eliminating matrix effects with liquid chromatography–mass spectrometry (Lc–ms)

In the process of a liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay development for two antimicrobial drugs using their stable isotope-labeled (SIL) analogues as internal standards to correct for matrix–ion suppression effects, an unusually large scatter of data was observed. A systematic observation revealed that the analytes and their SIL analogues (internal standards) were not completely coeluted; therefore, the analytes were not experiencing matrix effects similarly. When a column with lower resolution ability was used to achieve complete overlapping of the analyte and internal standard peaks, the scatter of LC–MS/MS data was minimized, indicating that the maximum correction of matrix effects by internal standards occurs when they are completely coeluted with the analytes. This study highlights the importance of ensuring complete overlapping of analyte and internal standard peaks in eliminating matrix effects when using SIL analogues as internal standards in LC–MS.